T cell receptors recognizing mhc class ii-restricted mage-a3

ABSTRACT

The invention provides an isolated or purified T-cell receptor (TCR) having antigenic specificity for MHC Class II-restricted MAGE-A3. The invention further provides related polypeptides and proteins, as well as related nucleic acids, recombinant expression vectors, host cells, and populations of cells. Further provided by the invention are antibodies, or an antigen binding portion thereof, and pharmaceutical compositions relating to the TCRs of the invention. Methods of detecting the presence of cancer in a host and methods of treating or preventing cancer in a mammal are further provided by the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 16/812,845, filed Mar. 9, 2020, which is a divisional of U.S.application Ser. No. 15/848,344, filed Dec. 20, 2017, which issued asU.S. Pat. No. 10,611,815, which is a divisional of U.S. application Ser.No. 14/427,671, which issued as U.S. Pat. No. 9,879,065, which is a U.S.National Phase of International Patent Application No.PCT/US2013/059608, filed Sep. 13, 2013, which claims the benefit of U.S.Provisional Application No. 61/701,056, filed on Sep. 14, 2012, each ofwhich is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under project numberZIABC010984 by the National Institutes of Health, National CancerInstitute. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 100,168 Byte XML file named “764792.XML,”dated Sep. 28, 2022.

BACKGROUND OF THE INVENTION

Adoptive cell therapy (ACT) involves the transfer of reactive T cellsinto patients, including the transfer of tumor-reactive T cells intocancer patients. Adoptive cell therapy using T-cells that target humanleukocyte antigen (HLA)-A*02 restricted T-cell epitopes has beensuccessful in causing the regression of tumors in some patients.However, patients that lack HLA-A*02 expression cannot be treated withT-cells that target HLA-A*02 restricted T-cell epitopes. Such alimitation creates an obstacle to the widespread application of adoptivecell therapy. Accordingly, there exists a need for improvedimmunological compositions and methods for treating cancer.

BRIEF SUMMARY OF THE INVENTION

An embodiment of the invention provides an isolated or purified T-cellreceptor (TCR), and functional portions and functional variants thereof,having antigenic specificity for MAGE-A3₂₄₃₋₂₅₈ and MAGE-A6.

Another embodiment of the invention provides an isolated or purified TCRcomprising (a) SEQ ID NOs: 3-8 or (b) SEQ ID NOs: 21-22, or a functionalvariant of (a) or (b), wherein the functional variant comprises (a) or(b) with at least one amino acid substitution in any one or more of (a)or any one or more of (b), and the functional variant has antigenicspecificity for MAGE-A3 in the context of HLA-DPβ1*04.

The invention further provides related polypeptides and proteins, aswell as related nucleic acids, recombinant expression vectors, hostcells, and populations of cells. Further provided by the invention areantibodies, or antigen binding portions thereof, and pharmaceuticalcompositions relating to the TCRs (including functional portions andfunctional variants thereof) of the invention.

Methods of detecting the presence of cancer in a mammal and methods oftreating or preventing cancer in a mammal are further provided by theinvention. The inventive method of detecting the presence of cancer in amammal comprises (i) contacting a sample comprising cells of the cancerwith any of the inventive TCRs (including functional portions andfunctional variants thereof), polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of host cells,or antibodies, or antigen binding portions thereof, described herein,thereby forming a complex, and (ii) detecting the complex, whereindetection of the complex is indicative of the presence of cancer in themammal.

The inventive method of treating or preventing cancer in a mammalcomprises administering to the mammal any of the TCRs (includingfunctional portions and functional variants thereof), polypeptides, orproteins described herein, any nucleic acid or recombinant expressionvector comprising a nucleotide sequence encoding any of the TCRs(including functional portions and functional variants thereof),polypeptides, proteins described herein, or any host cell or populationof host cells comprising a recombinant vector which encodes any of theTCRs (including functional portions and functional variants thereof),polypeptides, or proteins described herein, in an amount effective totreat or prevent cancer in the mammal.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIGS. 1A and 1B are bar graphs showing interferon (IFN)-gamma secretion(pg/ml) by CD4+ T cells from first (1A) and second (1B) donors inresponse to co-culture with 293-CIITA target cells untransfected(292-CIITA) or transfected with full length NY-ESO-1(293-CIITA-NY-ESO-1) protein, MAGE-A1 protein (293-CIITA-MAGE A1),MAGE-A3 protein (293-CIITA-MAGE-A3), MAGE-A6 protein(293-CIITA-MAGE-A6), or MAGE A12 protein (293-CIITA-MAGE-A12). The Tcells were untransduced (UT) or transduced with F5 (anti-MART-1) TCR,R12C9 TCR, or 6F9 TCR.

FIG. 2A is a bar graph showing IFN-gamma secretion (pg/ml) by T cellsfrom a human donor that were untransduced or transduced with 6F9 TCR orF5 TCR in response to co-culture with 526-CIITA cells that wereuntreated or treated with anti-MAGE-A3 siRNA or anti-MART-1 siRNA.

FIG. 2B is a bar graph showing IFN-gamma secretion (pg/ml) by CD4+ Tcells from a human donor that were untransduced or transduced with 6F9TCR in response to co-culture with H1299-CIITA cells that were untreatedor treated with anti-MAGE-A3 siRNA or anti-MART-1 siRNA.

FIG. 3 is a bar graph showing IFN-gamma secretion (pg/ml) by6F9-transduced PBL cultured alone (T cells only) or co-cultured with3071 cells, 3071-CIITA cells, 397 cells, 397-CIITA cells, 2630 cells,2630-CIITA cells, 2984 cells, or 2984-CIITA cells.

FIG. 4 is a bar graph showing IFN-gamma secretion (pg/ml) by CD4+enriched PBL that were transduced with 6F9 TCR or untransduced uponculture alone (T cell only) or in response to co-culture with untreatedH1299-CIITA cells, H1299-CIITA transfected with anti-HLA-DP oranti-HLA-DR siRNA, untreated 526-CIITA cells, or 526-CIITA transfectedwith anti-HLA-DP or anti-HLA-DR siRNA.

FIG. 5 is a bar graph showing IFN-gamma (pg/ml) secretion by PBL thatwere untransduced or transduced with wild-type (wt) 6F9 TCR or one ofeach of eight substituted TCRs: a1 (alpha chain S116A), a2 (alpha chainS117A), a3 (alpha chain G118A), a4 (alpha chain T119A), b1 (beta chainR115A), b2 (beta chain T116A), b3 (beta chain G117A), or b4 (beta chainP118A) upon culture alone (T cell only; unshaded bars) or co-culturewith 624-CIITA (checkered bars), 526-CIITA (right crosshatched bars),1359-CIITA (horizontal striped bars), H1299-CIITA (left crosshatchedbars), or 1764-CIITA (vertical striped bars).

FIG. 6 is a bar graph showing IFN-gamma (pg/ml) secretion by CD4+enriched PBL that were untransduced or transduced with wild-type (wt)6F9 TCR or one of each of three substituted TCRs: a1 (alpha chainS116A), a2 (alpha chain S117A), or b2 (beta chain T116A) upon culturealone (T cell only; unshaded bars) or co-culture with 624-CIITA(checkered bars), 526-CIITA (right crosshatched bars), 1359-CIITA(horizontal striped bars), H1299-CIITA (left crosshatched bars), or1764-CIITA (vertical striped bars).

FIG. 7 is a bar graph showing IFN-gamma (pg/ml) secretion by PBL thatwere untransduced or transduced with wild-type (wt) 6F9 TCR or one ofeach of ten substituted TCRs: a1 (alpha chain S116A), a2 (alpha chainS117A), a1-1 (alpha chain S116L), a1-2 (alpha chain S116I), a1-3 (alphachain S116V), a1-4 (alpha chain S116M), a2-1 (alpha chain S117L), a2-2(alpha chain S1171), a2-3 (alpha chain S117V), or a2-4 (alpha chainS117M) upon culture alone or (T cell only; unshaded bars) or co-culturewith 624-CIITA (right crosshatched bars), 526-CIITA (vertical stripedbars), 1359-CIITA (horizontal striped bars), H1299-CIITA (leftcrosshatched bars), or 1764-CIITA (black bars).

FIG. 8 is a bar graph showing IFN-gamma (pg/ml) secretion by PBL thatwere untransduced (checkered bars) or transduced with wild-type (wt) 6F9TCR (horizontal striped bar) or 6F9mC TCR (SEQ ID NOs: 27 and 28) (leftcrosshatched bars) upon culture alone (T cells only) or co-culture with624-CIITA, 1300-CIITA, 526-CIITA, 1359-CIITA, H1299-CIITA, 397-CIITA,2630-CIITA, 2984-CIITA, 3071-CIITA, or 1764-CIITA cells.

FIGS. 9A and 9B are bar graphs showing IFN-gamma (pg/ml) secretion byCD4+ (9A) or CD8+ (9B) enriched PBL that were untransduced (checkeredbars) or transduced with wild-type (wt) 6F9 TCR (horizontal striped bar)or 6F9mC TCR (SEQ ID NOs: 27 and 28) (left crosshatched bars) uponculture alone (T cells only) or co-culture with 624-CIITA, SK37-CIITA,526-CIITA, 1359-CIITA, H1299-CIITA, 397-CIITA, 2630-CIITA, 2984-CIITA,3071-CIITA, or 1764-CIITA cells.

FIG. 10A is a bar graph showing IFN-gamma secretion by PBL that wereuntransduced (UT; unshaded bars) or transduced with R12C9 TCR (greybars) or 6F9 TCR (black bars) upon culture alone (none) or co-culturewith 293-CIITA transfectants that were transfected with full lengthNY-ESO-1 protein, MAGE-A1 protein, MAGE-A3 protein, MAGE-A6 protein,MAGE-A12 protein, or 293-CIITA cells that were pulsed with MAGE-A3₂₄₃₋₂₅₈ peptide or MAGE-A3 protein.

FIG. 10B is a bar graph showing IFN-gamma secretion by PBL that wereuntransduced (UT; black bars) or transduced with 6F9 TCR (grey bars)upon culture alone (T cell alone) or co-culture with non-small cell lungcancer (NSCLC) cell line H11299 or melanoma cell line 526 mel, 624 mel,or 1359 mel.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated or purified T cell receptor (TCR),and functional portions and functional variants thereof, havingantigenic specificity for MAGE-A3, wherein the TCR recognizes MAGE-A3 inthe context of HLA-DPβ1*04. In an embodiment of the invention, theisolated or purified TCR has antigenic specificity for MAGE-A3₂₄₃₋₂₅₈and MAGE-A6.

MAGE-A3 and MAGE-A6 are members of the MAGE-A family of twelvehomologous proteins, also including MAGE-A1, MAGE-A2, MAGE-A4, MAGE-A5,MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, and MAGE-A12. The MAGE-Aproteins are cancer testis antigens (CTA), which are expressed only intumor cells and non-MHC expressing germ cells of the testis andplacenta. MAGE-A proteins are expressed in a variety of human cancersincluding, but not limited to, melanoma, breast cancer, leukemia,thyroid cancer, gastric cancer, pancreatic cancer, liver cancer (e.g.,hepatocellular carcinoma), lung cancer (e.g., non-small cell lungcarcinoma), ovarian cancer, multiple myeloma, esophageal cancer, kidneycancer, head cancers (e.g., squamous cell carcinoma), neck cancers(e.g., squamous cell carcinoma), prostate cancer, synovial cell sarcoma,and urothelial cancer.

The TCRs (including functional portions and functional variants thereof)of the invention provide many advantages, including when used foradoptive cell transfer. For example, by targeting MAGE-A3 that ispresented in the context of HLA-DPβ1*04, the inventive TCRs (includingfunctional portions and functional variants thereof) make it possible totreat patients who are unable to be treated using TCRs that target MAGEantigens that are presented in the context of other HLA molecules, e.g.,HLA-A*02, HLA-A*01, or HLA-C*07. HLA-DPβ1*04 is a highly prevalentallele that is expressed by from about 70% to about 80% of the cancerpatient population. Accordingly, the inventive TCRs (includingfunctional portions and functional variants thereof) advantageouslygreatly expand the patient population that can be treated. Additionally,without being bound by a particular theory, it is believed that becauseMAGE-A3 and/or MAGE-A6 are expressed by cells of multiple cancer types,the inventive TCRs (including functional portions and functionalvariants thereof) advantageously provide the ability to destroy cells ofmultiple types of cancer and, accordingly, treat or prevent multipletypes of cancer. Additionally, without being bound to a particulartheory, it is believed that because the MAGE-A proteins are cancertestis antigens that are expressed only in tumor cells and non-MHCexpressing germ cells of the testis and placenta, the inventive TCRs(including functional portions and functional variants thereof)advantageously target the destruction of cancer cells while minimizingor eliminating the destruction of normal, non-cancerous cells, therebyreducing, for example, by minimizing or eliminating, toxicity.

The phrase “antigenic specificity” as used herein means that the TCR canspecifically bind to and immunologically recognize MAGE-A3 and/orMAGE-A6 with high avidity. For example, a TCR may be considered to have“antigenic specificity” for MAGE-A3 and/or MAGE-A6 if T cells expressingthe TCR secrete at least about 200 pg/ml or more (e.g., 200 pg/ml ormore, 300 pg/ml or more, 400 pg/ml or more, 500 pg/ml or more, 600 pg/mlor more, 700 pg/ml or more, 1000 pg/ml or more, 5,000 pg/ml or more,7,000 pg/ml or more, 10,000 pg/ml or more, or 20,000 pg/ml or more) ofIFN-γ upon co-culture with antigen-negative HLA-DPβ1*04+ target cellspulsed with a low concentration of MAGE-A3 and/or MAGE-A6 peptide (e.g.,about 0.05 ng/ml to about 5 ng/ml, 0.05 ng/ml, 0.1 ng/ml, 0.5 ng/ml, 1ng/ml, or 5 ng/ml). Alternatively or additionally, a TCR may beconsidered to have “antigenic specificity” for MAGE-A3 and/or MAGE-A6 ifT cells expressing the TCR secrete at least twice as much IFN-γ as theuntransduced PBL background level of IFN-γ upon co-culture withantigen-negative HLA-DPβ1*04+ target cells pulsed with a lowconcentration of MAGE-A3 and/or MAGE-A6 peptide. The inventive TCRs(including functional portions and functional variants thereof) may alsosecrete IFN-γ upon co-culture with antigen-negative HLA-DPβ1*04+ targetcells pulsed with higher concentrations of MAGE-A3 and/or MAGE-A6peptide.

An embodiment of the invention provides a TCR (including functionalportions and variants thereof) with antigenic specificity for anyMAGE-A3 protein, polypeptide or peptide. The inventive TCR (includingfunctional portions and functional variants thereof) may have antigenicspecificity for a MAGE-A3 protein comprising, consisting of, orconsisting essentially of, SEQ ID NO: 1. In a preferred embodiment ofthe invention, the TCR (including functional portions and variantsthereof) has antigenic specificity for a MAGE-A3₂₄₃₋₂₅₈ peptidecomprising, consisting of, or consisting essentially of,KKLLTQHFVQENYLEY (SEQ ID NO: 2).

The inventive TCRs (including functional portions and functionalvariants thereof) are able to recognize MAGE-A3 in a human leukocyteantigen (HLA)-DPβ1*04-dependent manner. “HLA-DPβ1*04-dependent manner,”as used herein, means that the TCR elicits an immune response uponbinding to a MAGE-A3 protein, polypeptide or peptide within the contextof an HLA-DPβ1*04 molecule. The inventive TCRs (including functionalportions and functional variants thereof) are able to recognize MAGE-A3that is presented by an HLA-DPβ1*04 molecule and may bind to theHLA-DPβ1*04 molecule in addition to MAGE-A3. Exemplary HLA-DPβ1*04molecules, in the context of which the inventive TCRs (includingfunctional portions and functional variants thereof) recognize MAGE-A3,include those encoded by the HLA-DPβ1*0401 and/or HLA-DPβ1*0402 alleles.

An embodiment of the invention provides a TCR (including functionalportions and variants thereof) with antigenic specificity for anyMAGE-A6 protein, polypeptide or peptide. The inventive TCR (includingfunctional portions and functional variants thereof) may have antigenicspecificity for a MAGE-A6 protein comprising, consisting of, orconsisting essentially of, SEQ ID NO: 45. In a preferred embodiment ofthe invention, the TCR (including functional portions and functionalvariants thereof) has antigenic specificity for a MAGE-A6₂₄₃₋₂₅₈ peptidecomprising, consisting of, or consisting essentially of,KKLLTQYFVQENYLEY (SEQ ID NO: 46).

The invention provides a TCR comprising two polypeptides (i.e.,polypeptide chains), such as an alpha (α) chain of a TCR, a beta (β)chain of a TCR, a gamma (γ) chain of a TCR, a delta (δ) chain of a TCR,or a combination thereof. The polypeptides of the inventive TCR cancomprise any amino acid sequence, provided that the TCR has antigenicspecificity for MAGE-A3 in the context of HLA-DPβ1*04.

In an embodiment of the invention, the TCR comprises two polypeptidechains, each of which comprises a variable region comprising acomplementarity determining region (CDR)1, a CDR2, and a CDR3 of a TCR.In an embodiment of the invention, the TCR comprises a first polypeptidechain comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:3 or 13 (CDR1 of a chain), a CDR2 comprising the amino acid sequence ofSEQ ID NO: 4 or 14 (CDR2 of a chain), and a CDR3 comprising the aminoacid sequence of SEQ ID NO: 5 or 15 (CDR3 of a chain), and a secondpolypeptide chain comprising a CDR1 comprising the amino acid sequenceof SEQ ID NO: 6 or 16 (CDR1 of β chain), a CDR2 comprising the aminoacid sequence of SEQ ID NO: 7 or 17 (CDR2 of β chain), and a CDR3comprising the amino acid sequence of SEQ ID NO: 8 or 18 (CDR3 of βchain). In this regard, the inventive TCR can comprise any one or moreof the amino acid sequences selected from the group consisting of anyone or more of SEQ ID NOs: 3-5, 6-8, 13-15, and 16-18. Preferably theTCR comprises the amino acid sequences of SEQ ID NOs: 3-8 or 13-18. Morepreferably the TCR comprises the amino acid sequences of SEQ ID NOs:3-8.

Alternatively or additionally, the TCR can comprise an amino acidsequence of a variable region of a TCR comprising the CDRs set forthabove. In this regard, the TCR can comprise the amino acid sequence ofSEQ ID NO: 9 or 19 (the variable region of an α chain) or 10 or 20 (thevariable region of a β chain), both SEQ ID NOs: 9 and 10 or both SEQ IDNOs: 19 and 20. Preferably, the inventive TCR comprises the amino acidsequences of both SEQ ID NOs: 9 and 10.

Alternatively or additionally, the TCR can comprise an α chain of a TCRand a β chain of a TCR. Each of the α chain and β chain of the inventiveTCR can independently comprise any amino acid sequence. Preferably, theα chain comprises the variable region of an α chain as set forth above.In this regard, the inventive TCR can comprise the amino acid sequenceof SEQ ID NO: 11 or 21. An α chain of this type can be paired with any βchain of a TCR. Preferably, the β chain of the inventive TCR comprisesthe variable region of a β chain as set forth above. In this regard, theinventive TCR can comprise the amino acid sequence of SEQ ID NO: 12 or22. The inventive TCR, therefore, can comprise the amino acid sequenceof SEQ ID NO: 11, 12, 21, or 22, both SEQ ID NOs: 11 and 12 or both SEQID NOs: 21 and 22. Preferably, the inventive TCR comprises the aminoacid sequences of both SEQ ID NOs: 11 and 12.

Included in the scope of the invention are functional variants of theinventive TCRs described herein. The term “functional variant” as usedherein refers to a TCR, polypeptide, or protein having substantial orsignificant sequence identity or similarity to a parent TCR,polypeptide, or protein, which functional variant retains the biologicalactivity of the TCR, polypeptide, or protein of which it is a variant.Functional variants encompass, for example, those variants of the TCR,polypeptide, or protein described herein (the parent TCR, polypeptide,or protein) that retain the ability to specifically bind to MAGE-A3and/or MAGE-A6 for which the parent TCR has antigenic specificity or towhich the parent polypeptide or protein specifically binds, to a similarextent, the same extent, or to a higher extent, as the parent TCR,polypeptide, or protein. In reference to the parent TCR, polypeptide, orprotein, the functional variant can, for instance, be at least about30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical inamino acid sequence to the parent TCR, polypeptide, or protein.

The functional variant can, for example, comprise the amino acidsequence of the parent TCR, polypeptide, or protein with at least oneconservative amino acid substitution. Conservative amino acidsubstitutions are known in the art, and include amino acid substitutionsin which one amino acid having certain physical and/or chemicalproperties is exchanged for another amino acid that has the samechemical or physical properties. For instance, the conservative aminoacid substitution can be an acidic amino acid substituted for anotheracidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar sidechain substituted for another amino acid with a nonpolar side chain(e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basicamino acid substituted for another basic amino acid (Lys, Arg, etc.), anamino acid with a polar side chain substituted for another amino acidwith a polar side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.

Alternatively or additionally, the functional variants can comprise theamino acid sequence of the parent TCR, polypeptide, or protein with atleast one non-conservative amino acid substitution. In this case, it ispreferable for the non-conservative amino acid substitution to notinterfere with or inhibit the biological activity of the functionalvariant. Preferably, the non-conservative amino acid substitutionenhances the biological activity of the functional variant, such thatthe biological activity of the functional variant is increased ascompared to the parent TCR, polypeptide, or protein.

In this regard, an embodiment of the invention provides an isolated orpurified TCR comprising (a) SEQ ID NOs: 3-8, (b) SEQ ID NOs: 21-22, or afunctional variant of (a) or (b), wherein the functional variantcomprises (a) or (b) with at least one amino acid substitution in anyone or more of (a) or any one or more of (b), and the functional varianthas antigenic specificity for MAGE-A3 in the context of HLA-DPβ1*04.Preferably, the amino acid substitution is located in a CDR3 region ofthe alpha or beta chain, preferably in the CDR3 region of the alphachain. In some embodiments, the functional variant (or functionalportions thereof) provide an increased reactivity against MAGE-A3 ascompared to the parent TCR amino acid sequence. In general, thesubstituted α chain amino acid sequences SEQ ID NOs: 29, 31, and 33correspond with all or portions of the native, unsubstituted SEQ ID NO:11 (TCR α chain), with SEQ ID NOs: 29, 31, and 33 having at least onesubstitution when compared to SEQ ID NO: 11. Preferably, one or more ofthe native Ser116, Ser117, Gly118, and Thr119 is substituted. Likewise,the substituted 13 chain amino acid sequences SEQ ID NOs: 30, 32, and 34correspond with all or portions of the native, unsubstituted SEQ ID NO:12 (TCR β chain), with SEQ ID NOs: 30, 32, and 34 having at least onesubstitution when compared to SEQ ID NO: 12. Preferably, one or more ofthe native Arg115, Thr116, Gly117, and Pro118 is substituted.

In particular, the invention provides a functional variant of a TCRcomprising (i) SEQ ID NO: 29, wherein Xaa4 is Ser, Ala, Leu, Ile, Val,or Met; Xaa5 is Ser, Ala, Leu, Ile, Val, or Met; Xaa6 is Gly, Ala, Leu,Ile, Val, or Met; and Xaa7 is Thr, Ala, Leu, Ile, Val, or Met and/or(ii) SEQ ID NO: 30, wherein Xaa4 is Arg, Ala, Leu, Ile, Val, or Met;Xaa5 is Thr, Ala, Leu, Ile, Val, or Met; Xaa6 is Gly, Ala, Leu, Ile,Val, or Met; and Xaa7 is Pro, Ala, Leu, Ile, Val, or Met. SEQ ID NO: 29generally corresponds to positions 113-123 of the native, unsubstitutedSEQ ID NO: 11 with the exception that in SEQ ID NO: 29, one or more ofSer4, Ser5, Gly6, and Thr7 is substituted. Preferably, the functionalvariant comprises (a) SEQ ID NO: 29, wherein Xaa4 is Ala, Xaa5 is Ser,Xaa6 is Gly, and Xaa7 is Thr, or (b) SEQ ID NO: 29, wherein Xaa4 is Ser,Xaa5 is Ala, Xaa6 is Gly, and Xaa7 is Thr. Although in some embodiments,SEQ ID NO: 29 may comprise wild-type CDR3α SEQ ID NO: 5, preferably, SEQID NO: 29 does not comprise SEQ ID NO: 5. SEQ ID NO: 30 generallycorresponds to positions 112-126 of the native, unsubstituted SEQ ID NO:12 with the exception that in SEQ ID NO: 30, one or more of Arg4, Thr5,Gly6, and Pro7 is substituted. Although in some embodiments, SEQ ID NO:30 may comprise wild-type CDR3β SEQ ID NO: 8, preferably, SEQ ID NO: 30does not comprise SEQ ID NO: 8.

The invention also provides a functional variant of a TCR comprising (i)SEQ ID NO: 31, wherein Xaa116 is Ser, Ala, Leu, Ile, Val, or Met; Xaa117is Ser, Ala, Leu, Ile, Val, or Met; Xaa118 is Gly, Ala, Leu, Ile, Val,or Met; and Xaa119 is Thr, Ala, Leu, Ile, Val, or Met; and/or (ii) SEQID NO: 32, wherein Xaa115 is Arg, Ala, Leu, Ile, Val, or Met; Xaa116 isThr, Ala, Leu, Ile, Val, or Met; Xaa117 is Gly, Ala, Leu, Ile, Val, orMet; and Xaa118 is Pro, Ala, Leu, Ile, Val, or Met. SEQ ID NO: 31generally corresponds to positions 1-134 of the native, unsubstitutedSEQ ID NO: 11 with the exception that in SEQ ID NO: 31, one or more ofone or more of Ser116, Ser117, Gly118, and Thr119 is substituted.Preferably, the functional variant comprises (a) SEQ ID NO: 31, whereinXaa116 is Ala, Xaa117 is Ser, Xaa118 is Gly, and Xaa119 is Thr, or (b)SEQ ID NO: 31, wherein Xaa116 is Ser, Xaa117 is Ala, Xaa118 is Gly, andXaa119 is Thr. Although in some embodiments, SEQ ID NO: 31 may comprisewild-type CDR3a SEQ ID NO: 5, preferably, SEQ ID NO: 31 does notcomprise SEQ ID NO: 5. SEQ ID NO: 32 generally corresponds to positions1-137 of the native, unsubstituted SEQ ID NO: 12 with the exception thatin SEQ ID NO: 32, one or more of one or more of Arg115, Thr116, Gly117,and Pro118 is substituted. Although in some embodiments, SEQ ID NO: 32may comprise wild-type CDR3β SEQ ID NO: 8, preferably, SEQ ID NO: 32does not comprise SEQ ID NO: 8.

Also provided by the invention is functional variant of a TCR comprising(i) SEQ ID NO: 33, wherein Xaa116 is Ser, Ala, Leu, Ile, Val, or Met;Xaa117 is Ser, Ala, Leu, Ile, Val, or Met; Xaa118 is Gly, Ala, Leu, Ile,Val, or Met; and Xaa119 is Thr, Ala, Leu, Ile, Val, or Met; and/or (ii)SEQ ID NO: 34, wherein Xaa115 is Arg, Ala, Leu, Ile, Val, or Met; Xaa116is Thr, Ala, Leu, Ile, Val, or Met; Xaa117 is Gly, Ala, Leu, Ile, Val,or Met; and Xaa118 is Pro, Ala, Leu, Ile, Val, or Met. SEQ ID NO: 33generally corresponds to positions 1-275 of the native, unsubstitutedSEQ ID NO: 11 with the exception that in SEQ ID NO: 33, one or more ofone or more of Ser116, Ser117, Gly118, and Thr119 is substituted.Preferably, the functional variant comprises (a) SEQ ID NO: 33, whereinXaa116 is Ala, Xaa117 is Ser, Xaa118 is Gly, and Xaa119 is Thr, or (b)SEQ ID NO: 33, wherein Xaa116 is Ser, Xaa117 is Ala, Xaa118 is Gly, andXaa119 is Thr. Although in some embodiments, SEQ ID NO: 33 may comprisewild-type CDR3a SEQ ID NO: 5, preferably, SEQ ID NO: 33 does notcomprise SEQ ID NO: 5. SEQ ID NO: 34 generally corresponds to positions1-313 of the native, unsubstituted SEQ ID NO: 12 with the exception thatin SEQ ID NO: 34, one or more of one or more of Arg115, Thr116, Gly117,and Pro118 is substituted. Although in some embodiments, SEQ ID NO: 34may comprise wild-type CDR3β SEQ ID NO: 8, preferably, SEQ ID NO: 34does not comprise SEQ ID NO: 8.

Like the TCRs of the invention, the functional variants described hereincomprise two polypeptide chains, each of which comprises a variableregion comprising a CDR1, a CDR2, and a CDR3 of a TCR. Preferably, thefirst polypeptide chain comprises a CDR1 comprising the amino acidsequence of SEQ ID NO: 3 (CDR1 of α chain), a CDR2 comprising the aminoacid sequence of SEQ ID NO: 4 (CDR2 of α chain), and a substituted CDR3comprising the amino acid sequence of SEQ ID NO: 29 (substituted CDR3 ofα chain), and the second polypeptide chain comprises a CDR1 comprisingthe amino acid sequence of SEQ ID NO: 6 (CDR1 of β chain), a CDR2comprising the amino acid sequence of SEQ ID NO: 7 (CDR2 of β chain),and a CDR3 comprising the amino acid sequence of SEQ ID NO: 8 (CDR3 of βchain). In another embodiment, the first polypeptide chain comprises aCDR1 comprising the amino acid sequence of SEQ ID NO: 3 (CDR1 of achain), a CDR2 comprising the amino acid sequence of SEQ ID NO: 4 (CDR2of a chain), and a CDR3 comprising the amino acid sequence of SEQ ID NO:5 (CDR3 of α chain), and the second polypeptide chain comprises a CDR1comprising the amino acid sequence of SEQ ID NO: 6 (CDR1 of β chain), aCDR2 comprising the amino acid sequence of SEQ ID NO: 7 (CDR2 of βchain), and a substituted CDR3 comprising the amino acid sequence of SEQID NO: 30 (substituted CDR3 of β chain). In this regard, the inventivefunctional variant of a TCR can comprise the amino acid sequencesselected from the group consisting of SEQ ID NOs: 3-5; SEQ ID NOs: 3-4and 29; SEQ ID NOs: 6-8; and SEQ ID NOs: 6-7 and 30. Preferably thefunctional variant of a TCR comprises the amino acid sequences of SEQ IDNOs: 3-4, 29, and 6-8; SEQ ID NOs: 3-7 and 30; or SEQ ID NOs: 3-4, 29,6-7, and 30. More preferably, the functional variant of a TCR comprisesthe amino acid sequences of SEQ ID NOs: 3-4, 29, and 6-8.

Alternatively or additionally, the functional variant of a TCR cancomprise a substituted amino acid sequence of a variable region of a TCRcomprising the CDRs set forth above. In this regard, the TCR cancomprise the substituted amino acid sequence of SEQ ID NO: 31 (thesubstituted variable region of an α chain), 10 (the variable region of aβ chain), both SEQ ID NOs: 31 and 10, the substituted amino acidsequence of SEQ ID NO: 32 (the substituted variable region of an βchain), 9 (the variable region of an α chain), both SEQ ID NOs: 9 and32, or both SEQ ID NOs: 31 and 32. Preferably, the inventive functionalvariant of a TCR comprises the amino acid sequences of SEQ ID NOs: 31and 10 or SEQ ID NOs: 32 and 9. More preferably, the inventivefunctional variant of a TCR comprises the amino acid sequences of SEQ IDNOs: 31 and 10.

Alternatively or additionally, the functional variant of a TCR cancomprise a substituted α chain of a TCR and a β chain of a TCR. Each ofthe α chain and β chain of the inventive TCR can independently compriseany amino acid sequence. Preferably, the substituted α chain comprises asubstituted variable region of an α chain as set forth above. In thisregard, the inventive substituted α chain of the TCR can comprise theamino acid sequence of SEQ ID NO: 33. An inventive substituted α chainof this type can be paired with any β chain of a TCR. Preferably, the βchain of the inventive TCR comprises the variable region of a β chain asset forth above. In this regard, the inventive TCR can comprise theamino acid sequence of SEQ ID NO: 12 or the substituted amino acidsequence SEQ ID NO: 34. An inventive substituted β chain of this typecan be paired with any α chain of a TCR. In this regard, the inventiveTCR can comprise the amino acid sequence of SEQ ID NO: 11 or 33. Theinventive functional variant of a TCR, therefore, can comprise the aminoacid sequence of SEQ ID NO: 11, 12, 33, 34, both SEQ ID NOs: 33 and 34;both SEQ ID NOs: 11 and 34; or both SEQ ID NOs: 12 and 33. Preferably,the inventive functional variant of a TCR comprises the amino acidsequences of SEQ ID NOs: 11 and 34 or SEQ ID NOs: 12 and 33. Morepreferably, the functional variant of a TCR comprises the amino acidsequences of SEQ ID NOs: 12 and 33.

In an embodiment of the invention, the TCR (or functional variantthereof) may comprise a human constant region. In this regard, the TCR(or functional variant thereof) can comprise a human constant regioncomprising SEQ ID NO: 23 or 35 (human constant region of an α chain),SEQ ID NO: 24 or 36 (human constant region of β chain), both SEQ ID NOs:23 and 24, or both SEQ ID NOs: 35 and 36. Preferably, the TCR (orfunctional variant thereof) comprises both SEQ ID NOs: 23 and 24.

In another embodiment of the invention, the TCR (or functional variantthereof) can comprise a human/mouse chimeric TCR (or functional variantthereof). In this regard, the TCR (or functional variant thereof) cancomprise a mouse constant region comprising SEQ ID NO: 25 (mouseconstant region of an α chain), SEQ ID NO: 26 (mouse constant region ofβ chain), or both SEQ ID NOs: 25 and 26. Preferably, the TCR (orfunctional variant thereof) comprises both SEQ ID NOs: 25 and 26.

The inventive human/mouse chimeric TCR (or functional variant orfunctional portion thereof) can comprise any of the CDRs set forthabove. In this regard, the inventive human/mouse chimeric TCR (orfunctional variant or functional portion thereof) can comprise the aminoacid sequences selected from the group consisting of SEQ ID NOs: 3-5;SEQ ID NOs: 13-15; SEQ ID NOs: 16-18; SEQ ID NOs: 3-4 and 29; SEQ IDNOs: 6-8; and SEQ ID NOs: 6-7 and 30. Preferably the human/mousechimeric TCR (or functional variant or functional portion thereof)comprises the amino acid sequences of SEQ ID NOs: 3-8; SEQ ID NOs:13-18; SEQ ID NOs: 3-4, 29, and 6-8; SEQ ID NOs: 3-7 and 30; or SEQ IDNOs: 3-4, 29, 6-7, and 30. More preferably, the human/mouse chimeric TCR(or functional variant or functional portion thereof) comprises theamino acid sequences of SEQ ID NOs: 3-4, 29, and 6-8 or SEQ ID NOs: 3-8.

Alternatively or additionally, the human/mouse chimeric TCR (orfunctional variant or functional portion thereof) can comprise any ofthe variable regions set forth above. In this regard, the inventivehuman/mouse chimeric TCR (or functional variant or functional portionthereof) can comprise the substituted amino acid sequence of SEQ ID NO:31 (the substituted variable region of an α chain), SEQ ID NO: 10 or 20(the variable region of a β chain), the substituted amino acid sequenceof SEQ ID NO: 32 (the substituted variable region of an β chain), SEQ IDNO: 9 or 19 (the variable region of an α chain), both SEQ ID NOs: 9 and32, both SEQ ID NOs: 31 and 32, both SEQ ID NOs: 31 and 10, both SEQ IDNOs: 9 and 10, or both SEQ ID NOs: 19 and 20. Preferably, the inventivehuman/mouse chimeric TCR (or functional variant or functional portionthereof) comprises the amino acid sequences of SEQ ID NOs: 31 and 10,SEQ ID NOs: 9 and 10, or SEQ ID NOs: 32 and 9. More preferably, theinventive functional variant or functional portion of a TCR comprisesthe amino acid sequences of SEQ ID NOs: 31 and 10 or SEQ ID NOs: 9 and10.

Alternatively or additionally, the human/mouse chimeric TCR (orfunctional variant or functional portion thereof) can comprise an αchain of a TCR (or functional variant or functional portion thereof) anda β chain of a TCR (or functional variant or functional portionthereof). Each of the α chain and β chain of the inventive human/mousechimeric TCR (or functional variant or functional portion thereof) canindependently comprise any amino acid sequence. Preferably, the α chaincomprises the variable region of an α chain as set forth above. In thisregard, the inventive human/mouse chimeric TCR (or functional variant orfunctional portion thereof) can comprise the amino acid sequence of SEQID NO: 27. An inventive human/mouse chimeric TCR (or functional variantor functional portion thereof) of this type can be paired with any βchain of a TCR (or functional variant or functional portion thereof).Preferably, the β chain of the inventive human/mouse chimeric TCR (orfunctional variant or functional portion thereof) comprises the variableregion of a β chain as set forth above. In this regard, the inventivehuman/mouse chimeric TCR (or functional variant or functional portionthereof) can comprise the amino acid sequence of SEQ ID NO: 28. Theinventive human/mouse chimeric TCR (or functional variant or functionalportion thereof), therefore, can comprise the amino acid sequence of SEQID NO: 27 or 28, or both SEQ ID NOs: 27 and 28. Preferably, theinventive TCR comprises the amino acid sequences of SEQ ID NOs: 27 and28.

Also provided by the invention is a polypeptide comprising a functionalportion of any of the TCRs or functional variants described herein. Theterm “polypeptide” as used herein includes oligopeptides and refers to asingle chain of amino acids connected by one or more peptide bonds.

With respect to the inventive polypeptides, the functional portion canbe any portion comprising contiguous amino acids of the TCR (orfunctional variant thereof) of which it is a part, provided that thefunctional portion specifically binds to MAGE-A3 and/or MAGE-A6. Theterm “functional portion” when used in reference to a TCR (or functionalvariant thereof) refers to any part or fragment of the TCR (orfunctional variant thereof) of the invention, which part or fragmentretains the biological activity of the TCR (or functional variantthereof) of which it is a part (the parent TCR or parent functionalvariant thereof). Functional portions encompass, for example, thoseparts of a TCR (or functional variant thereof) that retain the abilityto specifically bind to MAGE-A3 (e.g., in an HLA-DPβ1*04-dependentmanner) or MAGE-A6, or detect, treat, or prevent cancer, to a similarextent, the same extent, or to a higher extent, as the parent TCR (orfunctional variant thereof). In reference to the parent TCR (orfunctional variant thereof), the functional portion can comprise, forinstance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of theparent TCR (or functional variant thereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, which additionalamino acids are not found in the amino acid sequence of the parent TCRor functional variant thereof. Desirably, the additional amino acids donot interfere with the biological function of the functional portion,e.g., specifically binding to MAGE-A3 and/or MAGE-A6; and/or having theability to detect cancer, treat or prevent cancer, etc. More desirably,the additional amino acids enhance the biological activity, as comparedto the biological activity of the parent TCR or functional variantthereof.

The polypeptide can comprise a functional portion of either or both ofthe α and β chains of the TCRs or functional variant thereof of theinvention, such as a functional portion comprising one of more of CDR1,CDR2, and CDR3 of the variable region(s) of the α chain and/or β chainof a TCR or functional variant thereof of the invention. In this regard,the polypeptide can comprise a functional portion comprising the aminoacid sequence of SEQ ID NO: 3 or 13 (CDR1 of α chain), 4 or 14 (CDR2 ofα chain), 5, 15, or 29 (CDR3 of α chain), 6 or 16 (CDR1 of β chain), 7or 17 (CDR2 of β chain), 8, 18, or 30 (CDR3 of β chain), or acombination thereof. Preferably, the inventive polypeptide comprises afunctional portion comprising SEQ ID NOs: 3-5; 3-4 and 29; 6-8; 6-7 and30; 13-15; 16-18; all of SEQ ID NOs: 3-8; all of SEQ ID NOs: 13-18; allof SEQ ID NOs: 3-4, 29, and 6-8; all of SEQ ID NOs: 3-7 and 30; or allof SEQ ID NOs: 3-4, 29, 6-7, and 30. More preferably, the polypeptidecomprises a functional portion comprising the amino acid sequences ofall of SEQ ID NOs: 3-8 or all of SEQ ID NOs: 3-4, 29, and 6-8.

Alternatively or additionally, the inventive polypeptide can comprise,for instance, the variable region of the inventive TCR or functionalvariant thereof comprising a combination of the CDR regions set forthabove. In this regard, the polypeptide can comprise the amino acidsequence of SEQ ID NO: 9, 19, or 31 (the variable region of an α chain),SEQ ID NO: 10, 20, or 32 (the variable region of a β chain), both SEQ IDNOs: 9 and 10, both SEQ ID NOs: 19 and 20; both SEQ ID NOs: 31 and 32;both SEQ ID NOs: 9 and 32; or both SEQ ID NOs: 10 and 31. Preferably,the polypeptide comprises the amino acid sequences of both SEQ ID NOs: 9and 10 or both SEQ ID NOs: 10 and 31.

Alternatively or additionally, the inventive polypeptide can comprisethe entire length of an α or β chain of one of the TCRs or functionalvariant thereof described herein. In this regard, the inventivepolypeptide can comprise an amino acid sequence of SEQ ID NOs: 11, 12,21, 22, 27, 28, 33, or 34. Alternatively, the polypeptide of theinvention can comprise α and β chains of the TCRs or functional variantsthereof described herein. For example, the inventive polypeptide cancomprise the amino acid sequences of both SEQ ID NOs: 11 and 12, bothSEQ ID NOs: 21 and 22, both SEQ ID NOs: 33 and 34, both SEQ ID NOs: 11and 34, both SEQ ID NOs: 12 and 33, or both SEQ ID NOs: 27 and 28.Preferably, the polypeptide comprises the amino acid sequences of bothSEQ ID NOs: 11 and 12, both SEQ ID NOs: 33 and 12, or both SEQ ID NOs:27 and 28.

The invention further provides a protein comprising at least one of thepolypeptides described herein. By “protein” is meant a moleculecomprising one or more polypeptide chains.

In an embodiment, the protein of the invention can comprise a firstpolypeptide chain comprising the amino acid sequences of SEQ ID NOs:3-5, SEQ ID NOs: 13-15, or SEQ ID NOs: 3-4 and 29 and a secondpolypeptide chain comprising the amino acid sequence of SEQ ID NOs: 6-8,SEQ ID NOs: 16-18, or SEQ ID NOs: 6-7 and 30. Alternatively oradditionally, the protein of the invention can comprise a firstpolypeptide chain comprising the amino acid sequence of SEQ ID NO: 9,19, or 31 and a second polypeptide chain comprising the amino acidsequence of SEQ ID NO: 10, 20, or 32. The protein of the invention can,for example, comprise a first polypeptide chain comprising the aminoacid sequence of SEQ ID NO: 11, 21, 27, or 33 and a second polypeptidechain comprising the amino acid sequence of SEQ ID NO: 12, 22, 28, or34. In this instance, the protein of the invention can be a TCR.Alternatively, if, for example, the protein comprises a singlepolypeptide chain comprising SEQ ID NO: 11, 21, 27, or 33 and SEQ ID NO:12, 22, 28, or 34, or if the first and/or second polypeptide chain(s) ofthe protein further comprise(s) other amino acid sequences, e.g., anamino acid sequence encoding an immunoglobulin or a portion thereof,then the inventive protein can be a fusion protein. In this regard, theinvention also provides a fusion protein comprising at least one of theinventive polypeptides described herein along with at least one otherpolypeptide. The other polypeptide can exist as a separate polypeptideof the fusion protein, or can exist as a polypeptide, which is expressedin frame (in tandem) with one of the inventive polypeptides describedherein. The other polypeptide can encode any peptidic or proteinaceousmolecule, or a portion thereof, including, but not limited to animmunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1 molecule, e.g.,CD1a, CD1b, CD1c, CD1d, etc.

The fusion protein can comprise one or more copies of the inventivepolypeptide and/or one or more copies of the other polypeptide. Forinstance, the fusion protein can comprise 1, 2, 3, 4, 5, or more, copiesof the inventive polypeptide and/or of the other polypeptide. Suitablemethods of making fusion proteins are known in the art, and include, forexample, recombinant methods. See, for instance, Choi et al., Mol.Biotechnol. 31: 193-202 (2005).

In some embodiments of the invention, the TCRs (and functional portionsand functional variants thereof), polypeptides, and proteins of theinvention may be expressed as a single protein comprising a linkerpeptide linking the α chain and the β chain. In this regard, the TCRs(and functional variants and functional portions thereof), polypeptides,and proteins of the invention comprising SEQ ID NO: 11, 21, 27, or 33and SEQ ID NO: 12, 22, 28, or 34 may further comprise a linker peptide.The linker peptide may advantageously facilitate the expression of arecombinant TCR (including functional portions and functional variantsthereof), polypeptide, and/or protein in a host cell. Upon expression ofthe construct including the linker peptide by a host cell, the linkerpeptide may be cleaved, resulting in separated α and β chains.

The protein of the invention can be a recombinant antibody comprising atleast one of the inventive polypeptides described herein. As usedherein, “recombinant antibody” refers to a recombinant (e.g.,genetically engineered) protein comprising at least one of thepolypeptides of the invention and a polypeptide chain of an antibody, ora portion thereof. The polypeptide of an antibody, or portion thereof,can be a heavy chain, a light chain, a variable or constant region of aheavy or light chain, a single chain variable fragment (scFv), or an Fc,Fab, or F(ab)₂′ fragment of an antibody, etc. The polypeptide chain ofan antibody, or portion thereof, can exist as a separate polypeptide ofthe recombinant antibody. Alternatively, the polypeptide chain of anantibody, or portion thereof, can exist as a polypeptide, which isexpressed in frame (in tandem) with the polypeptide of the invention.The polypeptide of an antibody, or portion thereof, can be a polypeptideof any antibody or any antibody fragment, including any of theantibodies and antibody fragments described herein.

The TCR (or functional variant thereof), polypeptide, or protein canconsist essentially of the specified amino acid sequence or sequencesdescribed herein, such that other components of the TCR (or functionalvariant thereof), polypeptide, or protein, e.g., other amino acids, donot materially change the biological activity of the TCR (or functionalvariant thereof), polypeptide, or protein. In this regard, the inventiveTCR (or functional variant thereof), polypeptide, or protein can, forexample, consist essentially of the amino acid sequence of SEQ ID NO:11, 12, 21, 22, 27, 28, 33, and 34, both SEQ ID NOs: 11 and 12, both SEQID NOs: 21 and 22, both SEQ ID NOs: 27 and 28, both SEQ ID NOs: 33 and34, both SEQ ID NOs: 11 and 34, or both SEQ ID NOs: 12 and 33. Also, forinstance, the inventive TCRs (including functional variants thereof),polypeptides, or proteins can consist essentially of the amino acidsequence(s) of SEQ ID NO: 9, 10, 19, 20, 31, 32, both SEQ ID NOs: 9 and10, both SEQ ID NOs: 19 and 20, both SEQ ID NOs: 31 and 32, both SEQ IDNOs: 9 and 32, both SEQ ID NOs: 10 and 31. Furthermore, the inventiveTCRs (including functional variants thereof), polypeptides, or proteinscan consist essentially of the amino acid sequence of SEQ ID NO: 3 or 13(CDR1 of α chain), SEQ ID NO: 4 or 14 (CDR2 of α chain), SEQ ID NO: 5,15, or 29 (CDR3 of α chain), SEQ ID NO: 6 or 16 (CDR1 of β chain), SEQID NO: 7 or 17 (CDR2 of β chain), SEQ ID NO: 8, 18, or 30 (CDR3 of βchain), or any combination thereof, e.g., SEQ ID NOs: 3-5; 6-8; 3-8;13-15; 16-18; 13-18; 3-4 and 29; 6-7 and 30; 3-4, 29, and 6-8; 3-7 and30; or 3-4, 29, 6-7, and 30.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be of any length, i.e., can compriseany number of amino acids, provided that the TCRs, polypeptides, orproteins (or functional variants thereof) retain their biologicalactivity, e.g., the ability to specifically bind to MAGE-A3 and/orMAGE-A6; detect cancer in a mammal; or treat or prevent cancer in amammal, etc. For example, the polypeptide can be in the range of fromabout 50 to about 5000 amino acids long, such as 50, 70, 75, 100, 125,150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more aminoacids in length. In this regard, the polypeptides of the invention alsoinclude oligopeptides.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) of the invention can comprise syntheticamino acids in place of one or more naturally-occurring amino acids.Such synthetic amino acids are known in the art, and include, forexample, aminocyclohexane carboxylic acid, norleucine, α-aminon-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- andtrans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserineβ-hydroxyphenylalanine, phenylglycine, α-naphthylalanine,cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid,aminomalonic acid monoamide, N′-benzyl-N′-methyl-lysine,N′,N′-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexane carboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid,α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine,and α-tert-butylglycine.

The TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be glycosylated, amidated,carboxylated, phosphorylated, esterified, N-acylated, cyclized via,e.g., a disulfide bridge, or converted into an acid addition salt and/oroptionally dimerized or polymerized, or conjugated.

The TCR, polypeptide, and/or protein of the invention (includingfunctional variants thereof) can be obtained by methods known in theart. Suitable methods of de novo synthesizing polypeptides and proteinsare described in references, such as Chan et al., Fmoc Solid PhasePeptide Synthesis, Oxford University Press, Oxford, United Kingdom,2005; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker,Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford UniversityPress, Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Also,polypeptides and proteins can be recombinantly produced using thenucleic acids described herein using standard recombinant methods. See,for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; andAusubel et al., Current Protocols in Molecular Biology, GreenePublishing Associates and John Wiley & Sons, N Y, 1994. Further, some ofthe TCRs, polypeptides, and proteins of the invention (includingfunctional variants thereof) can be isolated and/or purified from asource, such as a plant, a bacterium, an insect, a mammal, e.g., a rat,a human, etc. Methods of isolation and purification are well-known inthe art. Alternatively, the TCRs, polypeptides, and/or proteinsdescribed herein (including functional variants thereof) can becommercially synthesized by companies, such as Synpep (Dublin, Calif.),Peptide Technologies Corp. (Gaithersburg, Md.), and Multiple PeptideSystems (San Diego, Calif.). In this respect, the inventive TCRs(including functional variants thereof), polypeptides, and proteins canbe synthetic, recombinant, isolated, and/or purified.

Included in the scope of the invention are conjugates, e.g.,bioconjugates, comprising any of the inventive TCRs, polypeptides, orproteins (including any of the functional variants thereof), nucleicacids, recombinant expression vectors, host cells, populations of hostcells, or antibodies, or antigen binding portions thereof. Conjugates,as well as methods of synthesizing conjugates in general, are known inthe art (See, for instance, Hudecz, F., Methods Mol. Biol. 298: 209-223(2005) and Kirin et al., Inorg Chem. 44(15): 5405-5415 (2005)).

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. It is generally preferred that thenucleic acid does not comprise any insertions, deletions, inversions,and/or substitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids can be constructed based on chemical synthesis and/orenzymatic ligation reactions using procedures known in the art. See, forexample, Sambrook et al., supra, and Ausubel et al., supra. For example,a nucleic acid can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed upon hybridization (e.g.,phosphorothioate derivatives and acridine substituted nucleotides).Examples of modified nucleotides that can be used to generate thenucleic acids include, but are not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N⁶-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-substitutedadenine, 7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acid can comprise any nucleotide sequence which encodes anyof the TCRs, polypeptides, proteins, or functional functional variantsthereof described herein. For example, the nucleic acid can comprise,consist, or consist essentially of any one or more of the nucleotidesequence SEQ ID NOs: 37-44.

The invention also provides a nucleic acid comprising a nucleotidesequence which is complementary to the nucleotide sequence of any of thenucleic acids described herein or a nucleotide sequence which hybridizesunder stringent conditions to the nucleotide sequence of any of thenucleic acids described herein.

The nucleotide sequence which hybridizes under stringent conditionspreferably hybridizes under high stringency conditions. By “highstringency conditions” is meant that the nucleotide sequencespecifically hybridizes to a target sequence (the nucleotide sequence ofany of the nucleic acids described herein) in an amount that isdetectably stronger than non-specific hybridization. High stringencyconditions include conditions which would distinguish a polynucleotidewith an exact complementary sequence, or one containing only a fewscattered mismatches from a random sequence that happened to have a fewsmall regions (e.g., 3-10 bases) that matched the nucleotide sequence.Such small regions of complementarily are more easily melted than afull-length complement of 14-17 or more bases, and high stringencyhybridization makes them easily distinguishable. Relatively highstringency conditions would include, for example, low salt and/or hightemperature conditions, such as provided by about 0.02-0.1 M NaCl or theequivalent, at temperatures of about 50-70° C. Such high stringencyconditions tolerate little, if any, mismatch between the nucleotidesequence and the template or target strand, and are particularlysuitable for detecting expression of any of the inventive TCRs(including functional portions and functional variants thereof). It isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide.

The invention also provides a nucleic acid comprising a nucleotidesequence that is at least about 70% or more, e.g., about 80%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identical to any of the nucleic acidsdescribed herein.

The nucleic acids of the invention can be incorporated into arecombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the nucleic acids ofthe invention. For purposes herein, the term “recombinant expressionvector” means a genetically-modified oligonucleotide or polynucleotideconstruct that permits the expression of an mRNA, protein, polypeptide,or peptide by a host cell, when the construct comprises a nucleotidesequence encoding the mRNA, protein, polypeptide, or peptide, and thevector is contacted with the cell under conditions sufficient to havethe mRNA, protein, polypeptide, or peptide expressed within the cell.The vectors of the invention are not naturally-occurring as a whole.However, parts of the vectors can be naturally-occurring. The inventiverecombinant expression vectors can comprise any type of nucleotides,including, but not limited to DNA and RNA, which can be single-strandedor double-stranded, synthesized or obtained in part from naturalsources, and which can contain natural, non-natural or alterednucleotides. The recombinant expression vectors can comprisenaturally-occurring, non-naturally-occurring internucleotide linkages,or both types of linkages. Preferably, the non-naturally occurring oraltered nucleotides or internucleotide linkages does not hinder thetranscription or replication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host cell. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGT10,λGT11, λZapII (Stratagene), λEMBL4, and λNM1149, also can be used.Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from ColEl, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

Desirably, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host cell (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector can include one or more marker genes,which allow for selection of transformed or transfected host cells.Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostcell to provide prototrophy, and the like. Suitable marker genes for theinventive expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or nonnativepromoter operably linked to the nucleotide sequence encoding the TCR,polypeptide, or protein (including functional variants thereof), or tothe nucleotide sequence which is complementary to or which hybridizes tothe nucleotide sequence encoding the TCR, polypeptide, or protein(including functional variants thereof). The selection of promoters,e.g., strong, weak, inducible, tissue-specific anddevelopmental-specific, is within the ordinary skill of the artisan.Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter can be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The inventive recombinant expression vectors can be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors can be made for constitutive expressionor for inducible expression. Further, the recombinant expression vectorscan be made to include a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene can be a genethat confers sensitivity to an agent, e.g., a drug, upon the cell inwhich the gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews,Springer, Caroline J. (Cancer Research UK Centre for Cancer Therapeuticsat the Institute of Cancer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase,and nitroreductase.

Another embodiment of the invention further provides a host cellcomprising any of the recombinant expression vectors described herein.As used herein, the term “host cell” refers to any type of cell that cancontain the inventive recombinant expression vector. The host cell canbe a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell can be acultured cell or a primary cell, i.e., isolated directly from anorganism, e.g., a human. The host cell can be an adherent cell or asuspended cell, i.e., a cell that grows in suspension. Suitable hostcells are known in the art and include, for instance, DH5α E. colicells, Chinese hamster ovarian cells, monkey VERO cells, COS cells,HEK293 cells, and the like. For purposes of amplifying or replicatingthe recombinant expression vector, the host cell is preferably aprokaryotic cell, e.g., a DH5α cell. For purposes of producing arecombinant TCR, polypeptide, or protein, the host cell is preferably amammalian cell. Most preferably, the host cell is a human cell. Whilethe host cell can be of any cell type, can originate from any type oftissue, and can be of any developmental stage, the host cell preferablyis a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclearcell (PBMC). More preferably, the host cell is a T cell.

For purposes herein, the T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. Preferably, the T cell is a human T cell. More preferably,the T cell is a T cell isolated from a human. The T cell can be any typeof T cell and can be of any developmental stage, including but notlimited to, CD4⁺/CD8⁺ double positive T cells, CD4⁺ helper T cells,e.g., Th₁ and Th₂ cells, CD4+ T cells, CD8⁺ T cells (e.g., cytotoxic Tcells), tumor infiltrating lymphocytes (TILs), memory T cells (e.g.,central memory T cells and effector memory T cells), naïve T cells, andthe like.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells can be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell (e.g., a T cell), which does not compriseany of the recombinant expression vectors, or a cell other than a Tcell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, ahepatocyte, an endothelial cell, an epithelial cells, a muscle cell, abrain cell, etc. Alternatively, the population of cells can be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also can be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In one embodiment of the invention, the population of cells is a clonalpopulation comprising host cells comprising a recombinant expressionvector as described herein.

The invention further provides an antibody, or antigen binding portionthereof, which specifically binds to a functional portion of any of theTCRs (or functional variant thereof) described herein. Preferably, thefunctional portion specifically binds to the cancer antigen, e.g., thefunctional portion comprising the amino acid sequence SEQ ID NO: 3 or 13(CDR1 of α chain), 4 or 14 (CDR2 of α chain), 5, 15, or 29 (CDR3 of αchain), 6 or 16 (CDR1 of β chain), 7 or 17 (CDR2 of β chain), 8, 18, or30 (CDR3 of β chain), SEQ ID NO: 9, 19, or 31 (variable region of αchain), SEQ ID NO: 10, 20, or 32 (variable region of β chain), or acombination thereof, e.g., 3-5; 6-8; 3-8; 13-15; 16-18; 13-18; 3-4 and29; 6-7 and 30; 3-4, 29, and 6-8; or 3-7 and 30; 3-4, 29, 6-7, and 30.More preferably, the functional portion comprises the amino acidsequences of SEQ ID NOs: 3-8 or SEQ ID NOs: 3-4, 29, and 6-8. In apreferred embodiment, the antibody, or antigen binding portion thereof,binds to an epitope which is formed by all 6 CDRs (CDR1-3 of the alphachain and CDR1-3 of the beta chain). The antibody can be any type ofimmunoglobulin that is known in the art. For instance, the antibody canbe of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody canbe monoclonal or polyclonal. The antibody can be a naturally-occurringantibody, e.g., an antibody isolated and/or purified from a mammal,e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.Alternatively, the antibody can be a genetically-engineered antibody,e.g., a humanized antibody or a chimeric antibody. The antibody can bein monomeric or polymeric form. Also, the antibody can have any level ofaffinity or avidity for the functional portion of the inventive TCR (orfunctional variant thereof). Desirably, the antibody is specific for thefunctional portion of the inventive TCR (or functional variantsthereof), such that there is minimal cross-reaction with other peptidesor proteins.

Methods of testing antibodies for the ability to bind to any functionalportion or functional variant of the inventive TCR are known in the artand include any antibody-antigen binding assay, such as, for example,radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation, andcompetitive inhibition assays (see, e.g., Janeway et al., infra, andU.S. Patent Application Publication No. 2002/0197266 A1).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing, New York,N.Y. (2001)). Alternatively, other methods, such as EBV-hybridomamethods (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984),and Roder et al., Methods Enzymol., 121, 140-67 (1986)), andbacteriophage vector expression systems (see, e.g., Huse et al.,Science, 246, 1275-81 (1989)) are known in the art. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1.

Phage display furthermore can be used to generate the antibody of theinvention. In this regard, phage libraries encoding antigen-bindingvariable (V) domains of antibodies can be generated using standardmolecular biology and recombinant DNA techniques (see, e.g., Sambrook etal. (eds.), Molecular Cloning, A Laboratory Manual, 3^(rd) Edition, ColdSpring Harbor Laboratory Press, New York (2001)). Phage encoding avariable region with the desired specificity are selected for specificbinding to the desired antigen, and a complete or partial antibody isreconstituted comprising the selected variable domain. Nucleic acidsequences encoding the reconstituted antibody are introduced into asuitable cell line, such as a myeloma cell used for hybridomaproduction, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in, for example, U.S. Pat. No. 5,639,641 and Pedersen et al.,J. Mol. Biol., 235, 959-973 (1994).

The invention also provides antigen binding portions of any of theantibodies described herein. The antigen binding portion can be anyportion that has at least one antigen binding site, such as Fab,F(ab′)₂, dsFv, sFv, diabodies, and triabodies.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)). Antibody fragments of theinvention, however, are not limited to these exemplary types of antibodyfragments.

Also, the antibody, or antigen binding portion thereof, can be modifiedto comprise a detectable label, such as, for instance, a radioisotope, afluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin(PE)), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase),and element particles (e.g., gold particles).

The inventive TCRs, polypeptides, proteins, (including functionalvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), and antibodies (including antigenbinding portions thereof), can be isolated and/or purified. The term“isolated” as used herein means having been removed from its naturalenvironment. The term “purified” as used herein means having beenincreased in purity, wherein “purity” is a relative term, and not to benecessarily construed as absolute purity. For example, the purity can beat least about 50%, can be greater than 60%, 70%, 80%, 90%, 95%, or canbe 100%.

The inventive TCRs, polypeptides, proteins (including functionalvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), and antibodies (including antigenbinding portions thereof), all of which are collectively referred to as“inventive TCR materials” hereinafter, can be formulated into acomposition, such as a pharmaceutical composition. In this regard, theinvention provides a pharmaceutical composition comprising any of theTCRs, polypeptides, proteins, functional portions, functional variants,nucleic acids, expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),and a pharmaceutically acceptable carrier. The inventive pharmaceuticalcompositions containing any of the inventive TCR materials can comprisemore than one inventive TCR material, e.g., a polypeptide and a nucleicacid, or two or more different TCRs (including functional portions andfunctional variants thereof). Alternatively, the pharmaceuticalcomposition can comprise an inventive TCR material in combination withanother pharmaceutically active agents or drugs, such as achemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin,cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine,hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine,vincristine, etc.

Preferably, the carrier is a pharmaceutically acceptable carrier. Withrespect to pharmaceutical compositions, the carrier can be any of thoseconventionally used for the particular inventive TCR material underconsideration. Such pharmaceutically acceptable carriers are well-knownto those skilled in the art and are readily available to the public. Itis preferred that the pharmaceutically acceptable carrier be one whichhas no detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularinventive TCR material, as well as by the particular method used toadminister the inventive TCR material. Accordingly, there are a varietyof suitable formulations of the pharmaceutical composition of theinvention. Suitable formulations may include any of those for oral,aerosol, parenteral, subcutaneous, intravenous, intramuscular,intraarterial, intrathecal, or interperitoneal administration. More thanone route can be used to administer the inventive TCR materials, and incertain instances, a particular route can provide a more immediate andmore effective response than another route.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously. When the inventive TCR material is a host cellexpressing the inventive TCR (or functional variant thereof), thepharmaceutically acceptable carrier for the cells for injection mayinclude any isotonic carrier such as, for example, normal saline (about0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott,Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5%dextrose in water, or Ringer's lactate. In an embodiment, thepharmaceutically acceptable carrier is supplemented with human serumalbumen.

In an embodiment of the invention, the pharmaceutical composition mayfurther comprise MHC Class I restricted TCRs, or polypeptides, proteins,nucleic acids, or recombinant expression vectors encoding MHC Class Irestricted TCRs, or host cells or populations of cells expressing MHCClass I restricted TCRs. Without being bound to a particular theory, itis believed that MHC Class I restricted CD8+ T cells augment thereactivity of MHC Class II restricted CD4+ T cells and enhance theability of the MHC Class II restricted CD4+ T cells to treat or preventcancer.

For purposes of the invention, the amount or dose (e.g., numbers ofcells when the inventive TCR material is one or more cells) of theinventive TCR material administered should be sufficient to effect,e.g., a therapeutic or prophylactic response, in the subject or animalover a reasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes of the invention, an assay, which comprises comparing theextent to which target cells are lysed or IFN-γ is secreted by T cellsexpressing the inventive TCR (or functional variant or functionalportion thereof), polypeptide, or protein upon administration of a givendose of such T cells to a mammal among a set of mammals of which is eachgiven a different dose of the T cells, could be used to determine astarting dose to be administered to a mammal. The extent to which targetcells are lysed or IFN-γ is secreted upon administration of a certaindose can be assayed by methods known in the art.

The dose of the inventive TCR material also will be determined by theexistence, nature and extent of any adverse side effects that mightaccompany the administration of a particular inventive TCR material.Typically, the attending physician will decide the dosage of theinventive TCR material with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, inventive TCR material to beadministered, route of administration, and the severity of the conditionbeing treated. In an embodiment in which the inventive TCR material is apopulation of cells, the number of cells administered per infusion mayvary, e.g., from about 1×10⁶ to about 1×10¹¹ cells or more.

One of ordinary skill in the art will readily appreciate that theinventive TCR materials of the invention can be modified in any numberof ways, such that the therapeutic or prophylactic efficacy of theinventive TCR materials is increased through the modification. Forinstance, the inventive TCR materials can be conjugated either directlyor indirectly through a bridge to a targeting moiety. The practice ofconjugating compounds, e.g., inventive TCR materials, to targetingmoieties is known in the art. See, for instance, Wadwa et al., J DrugTargeting 3: 111 (1995) and U.S. Pat. No. 5,087,616. The term “targetingmoiety” as used herein, refers to any molecule or agent thatspecifically recognizes and binds to a cell-surface receptor, such thatthe targeting moiety directs the delivery of the inventive TCR materialsto a population of cells on which surface the receptor is expressed.Targeting moieties include, but are not limited to, antibodies, orfragments thereof, peptides, hormones, growth factors, cytokines, andany other natural or non-natural ligands, which bind to cell surfacereceptors (e.g., Epithelial Growth Factor Receptor (EGFR), T-cellreceptor (TCR), B-cell receptor (BCR), CD28, Platelet-derived GrowthFactor Receptor (PDGF), nicotinic acetylcholine receptor (nAChR), etc.).The term “bridge” as used herein, refers to any agent or molecule thatlinks the inventive TCR materials to the targeting moiety. One ofordinary skill in the art recognizes that sites on the inventive TCRmaterials, which are not necessary for the function of the inventive TCRmaterials, are ideal sites for attaching a bridge and/or a targetingmoiety, provided that the bridge and/or targeting moiety, once attachedto the inventive TCR materials, do(es) not interfere with the functionof the inventive TCR materials, i.e., the ability to bind to MAGE-A3 orMAGE-A6; or to detect, treat, or prevent cancer.

It is contemplated that the inventive pharmaceutical compositions, TCRs(including functional variants thereof), polypeptides, proteins, nucleicacids, recombinant expression vectors, host cells, or populations ofcells can be used in methods of treating or preventing cancer. Withoutbeing bound to a particular theory, the inventive TCRs (and functionalvariants thereof) are believed to bind specifically to MAGE-A3 and/orMAGE-A6, such that the TCR (or related inventive polypeptide or proteinand functional variants thereof) when expressed by a cell is able tomediate an immune response against a target cell expressing MAGE-A3 orMAGE-A6. In this regard, the invention provides a method of treating orpreventing cancer in a mammal, comprising administering to the mammalany of the pharmaceutical compositions, TCRs (and functional variantsthereof), polypeptides, or proteins described herein, any nucleic acidor recombinant expression vector comprising a nucleotide sequenceencoding any of the TCRs (and functional variants thereof),polypeptides, proteins described herein, or any host cell or populationof cells comprising a recombinant vector which encodes any of the TCRs(and functional variants thereof), polypeptides, or proteins describedherein, in an amount effective to treat or prevent cancer in the mammal.

In an embodiment of the invention, the inventive methods of treating orpreventing cancer may further comprise co-administering MHC Class Irestricted TCRs, or polypeptides, proteins, nucleic acids, orrecombinant expression vectors encoding MHC Class I restricted TCRs, orhost cells or populations of cells expressing MHC Class I restrictedTCRs, to the mammal.

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof cancer in a mammal. Furthermore, the treatment or prevention providedby the inventive method can include treatment or prevention of one ormore conditions or symptoms of the disease, e.g., cancer, being treatedor prevented. Also, for purposes herein, “prevention” can encompassdelaying the onset of the disease, or a symptom or condition thereof.

Also provided is a method of detecting the presence of cancer in amammal. The method comprises (i) contacting a sample comprising cells ofthe cancer with any of the inventive TCRs (and functional variantsthereof), polypeptides, proteins, nucleic acids, recombinant expressionvectors, host cells, populations of cells, or antibodies, or antigenbinding portions thereof, described herein, thereby forming a complex,and detecting the complex, wherein detection of the complex isindicative of the presence of cancer in the mammal.

With respect to the inventive method of detecting cancer in a mammal,the sample of cells of the cancer can be a sample comprising wholecells, lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive TCRs (and functionalvariants thereof), polypeptides, proteins, nucleic acids, recombinantexpression vectors, host cells, populations of cells, or antibodies, orantigen binding portions thereof, described herein, can be labeled witha detectable label such as, for instance, a radioisotope, a fluorophore(e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme(e.g., alkaline phosphatase, horseradish peroxidase), and elementparticles (e.g., gold particles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the inventive methods, the cancer can be any cancer,including any of sarcomas (e.g., synovial sarcoma, osteogenic sarcoma,leiomyosarcoma uteri, and alveolar rhabdomyosarcoma), lymphomas (e.g.,Hodgkin lymphoma and non-Hodgkin lymphoma), hepatocellular carcinoma,glioma, head cancers (e.g., squamous cell carcinoma), neck cancers(e.g., squamous cell carcinoma), acute lymphocytic cancer, leukemias(e.g., acute myeloid leukemia and chronic lymphocytic leukemia), bonecancer, brain cancer, breast cancer, cancer of the anus, anal canal, oranorectum, cancer of the eye, cancer of the intrahepatic bile duct,cancer of the joints, cancer of the neck, gallbladder, or pleura, cancerof the nose, nasal cavity, or middle ear, cancer of the oral cavity,cancer of the vulva, chronic myeloid cancer, colon cancers (e.g., coloncarcinoma), esophageal cancer, cervical cancer, gastric cancer,gastrointestinal carcinoid tumor, hypopharynx cancer, larynx cancer,liver cancers (e.g., hepatocellular carcinoma), lung cancers (e.g.,non-small cell lung carcinoma), malignant mesothelioma, melanoma,multiple myeloma, nasopharynx cancer, ovarian cancer, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, kidney cancers (e.g., renal cell carcinoma),small intestine cancer, soft tissue cancer, stomach cancer, testicularcancer, thyroid cancer, and urothelial cancers (e.g., ureter cancer andurinary bladder cancer). Preferably, the cancer is melanoma, breastcancer, lung cancer, prostate cancer, synovial cell sarcoma, head andneck cancer, esophageal cancer, or ovarian cancer.

The mammal referred to in the inventive methods can be any mammal. Asused herein, the term “mammal” refers to any mammal, including, but notlimited to, mammals of the order Rodentia, such as mice and hamsters,and mammals of the order Logomorpha, such as rabbits. It is preferredthat the mammals are from the order Carnivora, including Felines (cats)and Canines (dogs). It is more preferred that the mammals are from theorder Artiodactyla, including Bovines (cows) and Swines (pigs) or of theorder Perssodactyla, including Equines (horses). It is most preferredthat the mammals are of the order Primates, Ceboids, or Simoids(monkeys) or of the order Anthropoids (humans and apes). An especiallypreferred mammal is the human.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1A

This example demonstrates the isolation of TCRs from T cell clones.

Anti-MAGE-A3₂₄₃₋₂₅₈ CD4+ effector clone R12C9 and anti-MAGE-A3₂₄₃₋₂₅₈Treg clone 6F9 was cultured with peptide (MAGE-A3₂₄₃₋₂₅₈)-pulsed EBV Bcells. Cytokine secretion, percentage of indicator cells suppressed,percentage of FOXP3+ Treg cells, and percentage of unmethylated FOXP3sequences were measured. Unmethylated FOXP3 intron 1 sequences areconsidered to be a marker for a stable Treg phenotype. The results forthe 6F9 and R12C9 clones are shown in Tables 1A and 1B.

TABLE 1A % Indicator Cells % Unmethylated Clone Suppressed % FOXP3+FOXP3 sequences 6F9 57 95 72 R12C9 0 8 2

TABLE 1B Cytokine Secretion (pg/25,000 cells) Clone IFN-γ IL-2 IL-10IL-4 IL-5 TNF-α 6F9 0 0 0 0 0 12 R12C9 922 46 479 8 28 422

Treg clones can inhibit the proliferation of indicator cells afterstimulation by an appropriate peptide. As shown in Table 1A, clone 6F9is a Treg clone.

A TCR comprising SEQ ID NOs: 21 and 22 was cloned from theanti-MAGE-A3₂₄₃₋₂₅₈ CD4+ effector clone R12C9 (“R12C9 TCR”). A TCRcomprising SEQ ID NOs: 11 and 12 was cloned from the anti-MAGE-A3₂₄₃₋₂₅₈Treg clone 6F9 (“6F9 TCR”).

Example 1B

This example demonstrates the transduction efficiency of PBMC transducedwith a nucleotide sequence encoding the 6F9 TCR or R12C9 TCR of Example1.

Transcripts encoding the TCR alpha and beta chains of R12C9 and 6F9 werelinked with sequences encoding a P2A self-cleaving peptide and clonedinto an MSGV1 retroviral vector. PBMC from three patients werestimulated with OKT3, transduced with transient retroviral supernatantson day two, and enriched for CD4+ T cells on day seven. The levels ofTCR expression were evaluated by staining transduced cells withanti-V(322 or V136.7, which detect the 6F9 or R12C9 TCR, respectively.Analysis of PBMC from patient 1 indicated that between 25 and 35% of theT cells were transduced with the individual TCRs and similartransduction levels were obtained with PBMC from patients 2 and 3.

Example 2

This example demonstrates that T cells transduced with nucleotidesequences encoding the anti-MAGE-A3₂₄₃₋₂₅₈ TCRs of Example 1 recognize293-class II, major histocompatibility complex, transactivator (CIITA)transfectants of MAGE-A3 and peptide-pulsed targets. This example alsodemonstrates that the 6F9 TCR recognizes 293-CIITA transfectants ofMAGE-A3 and MAGE-A6.

CD4+ enriched peripheral blood lymphocytes (PBL) from two human donorswere untransduced (UT) or transduced with F5 (anti-MART-1) TCR, R12C9TCR, or 6F9 TCR. The cells were cultured with 293-CIITA-transfectedtarget cells pulsed with MAGE-A3₂₄₃₋₂₅₈ (SEQ ID NO: 2) peptide. The293-CIITA cells are 293 cells transduced with CIITA, which is a humangene which encodes the class II, major histocompatibility complextransactivator protein. The results obtained with 6F9 and R12C9TCR-transduced cells are shown in Table 2 and FIG. 10A. PBL transducedby R12C9 TCR or 6F9 TCR recognized MAGE-A3₂₄₃₋₂₅₈ peptide-pulsedHLA-DP*0401+ target cells. Titration of the MAGE-A3₂₄₃₋₂₅₈ peptideindicated that CD4⁺ T cells transduced with the 6F9 or R12C9 TCRsreleased comparable levels of IFN-γ in response to targets pulsed with aminimum of between 0.001 and 0.01 mg/ml of the MAGE-A3:₂₄₃₋₂₅₈ peptide.The experiments were repeated using PBL from a third human donor andsimilar results were obtained.

TABLE 2 Donor 1 IFN-gamma (pg/ml) Peptide Untransduced 6F9 TCR (μg/ml)(UT) transduced 293-CIITA + MAGE- DP4+ 0.0001 2 328 A3₂₄₃₋₂₅₈ 0.001 17596 0.01 4 1609 0.1 2 7440 1 4 34800 10 0 52100 Donor 2 IFN-gamma(pg/ml) Peptide Untransduced 6F9 TCR (μg/ml) (UT) transduced 293-CIITA +MAGE- DP4+ 0.0001 28 110 A3₂₄₃₋₂₅₈ 0.001 30 323 0.01 37 1830 0.1 40 97601 44 55000 10 0 59050

293-CIITA target cells were transfected with DNA constructs (pCDNA3vector) encoding full-length MAGE-A3 protein or MAGE-A6 protein, whichdiffer at only a single position (249), or full-length MAGE-A1 proteinor MAGE-A12 protein. Untransduced and transduced PBL were co-culturedwith the transfected 293-CIITA cells and interferon (IFN) gammasecretion was measured. The results are shown in FIGS. 1A, 1B, and 10A.

As shown in FIGS. 1A, 1B, and 10A, although T cells transduced withR12C9 TCR or 6F9 TCR recognized peptide-pulsed targets, PBL transducedwith the 6F9 TCR were the most highly reactive to each of MAGE-A3 andMAGE-A6 293-CIITA transfectants. CD4+ T cells transduced with the 6F9but not the R12C9 TCR recognized HLA DP*0401⁺293-CIITA cells transfectedwith genes encoding MAGE-A3 or MAGE-A6, but not MAGE-A1 or A12.Comparison of amino acid sequences of the corresponding regions of theMAGE family members indicated that MAGE-A3 and MAGE-A6 only differed atone position (residue 249), whereas the other MAGE family membersdiffered from MAGE-A3 at two (MAGE-A12₂₄₃₋₂₅₈ (SEQ ID NO: 70)) or three(MAGE-A1₂₄₃₋₂₅₈ (SEQ ID NO: 71)) positions. In addition, CD4⁺ T cellstransduced with the 6F9 TCR but not the R12C9 TCR recognized theMAGE-A3⁺/HLA-DP*0401⁺ melanoma cell line 1359 mel-CIITA but failed torecognize the MAGE-A3⁺/HLA-DP*0401⁻ melanoma cell line 624 mel-CIITA.CD4⁺ T cells transduced with the R12C9 TCR failed to recognize either ofthe tested melanoma cell lines. Cells transduced with the MART-1reactive TCR DMF5 failed to recognize the transfected 293-CIITA cells orMAGE-A3:₂₄₃₋₂₅₈ pulsed target cells, but recognized the HLA-A*0201+ andMART-1+ cell line 624 mel-CIITA. The experiments were repeated using PBLfrom a third human donor and similar results were obtained. Because the6F9 TCR was obtained from a Treg clone, which are involved in thesuppression of immune activity, the reactivity of the 6F9 TCR wassurprising and unexpected. These results indicated that while CD4⁺ Tcells transduced with 6F9 or R12C9 recognized peptide pulsed targetcells, only cells transduced with the 6F9 TCR recognized transfectedtarget cells as well as MAGE-A3⁺ and HLA-DP*04⁺ tumor cells.

Example 3

This example demonstrates that 6F9-transduced PBLs show high reactivityto MAGE-A3 full-length protein processed and presented by HLA-DP4+ Bcells.

PBL from two human donors was untransduced or transduced with anucleotide sequence encoding the 6F9 TCR. The cells were co-culturedwith HLA-DP4+ B cells that had processed and presented full-lengthMAGE-A3 protein (SEQ ID NO: 1). The results are shown in Table 3 andFIG. 10A. As shown in Table 3 and FIG. 10A, the 6F9-transduced PBLs werehighly reactive to MAGE-A3 full-length protein processed and presentedby HLA-DP4+ B cells.

TABLE 3 Donor 1 IFN-γ (pg/ml) MAGE-A3 Untransduced 6F9 TCR DP4 (μg/ml)(UT) transduced B cells + MAGE-A3 + 10 360 23640 full length + 1 42012440 + 0.1 358 2360 + 0.01 362 580 + 0.001 343 427 + 0.0001 349 387 + 0343 405 B cells + NY-ESO-1 + 10 313 363 full length Donor 2 IFN-γ(pg/ml) MAGE-A3 Untransduced 6F9 TCR DP4 (μg/ml) (UT) transduced Bcells + MAGE-A3 + 10 2080 63100 full length + 1 1810 21270 + 0.1 13823590 + 0.01 1519 685 + 0.001 1297 470 + 0.0001 1568 542 + 0 1351 404 Bcells + NY-ESO-1 + 10 1549 530 full length

Example 4

This example demonstrates that 6F9 TCR-transduced PBLs are reactive totumor lines with endogenous class II presentation of MAGE-A3 protein.

PBL from two human donors were untransduced or transduced with anucleotide sequence encoding the 6F9 TCR or F5 TCR. The cells werecultured alone (T cell only) or co-cultured with 624-CIITA cells,526-CIITA cells, or H1299-CIITA cells (tumor cell lines transfected withCIITA). The results are shown in Table 4. As shown in Table 4, 6F9TCR-transduced PBLs were reactive to tumor lines with endogenous classII presentation of MAGE-A3 protein.

TABLE 4 IFN-gamma (pg/ml) Donor 1 Un- F5 6F9 DP4 MAGE A3 transducedtransduced transduced 624-CIITA − + 223 1483 238 526-CIITA + (DP40401) + 636 2360 1314 H1299- + (DP4 0401) + 284 243 4330 CIITA T-cellonly 131 45 112 IFN-gamma (pg/ml) Donor 2 Un- F5 6F9 DP4 MAGE A3transduced transduced transduced 624-CIITA − + 819 1435 153526-CIITA + + 117 2530 1339 H1299- + + 147 172 3630 CIITA T-cell only 6527 88

Example 5

This example demonstrates that the 6F9 TCR is MAGE-A3 specific.

PBL from a human donor were CD4+ enriched and the number of cells wasrapidly expanded on day 27. Cells were untransduced or transduced withF5 TCR or 6F9 TCR and co-cultured with 526-CIITA cells or H1299-CIITAcells alone or with anti-MAGE-A3 siRNA or anti-MART-1 siRNA. IFN-gammasecretion was measured. The results are shown in FIGS. 2A and 2B.

As shown in FIGS. 2A and 2B, the anti-MAGE-A3 siRNA reduced thereactivity of the 6F9-TCR transduced cells. Accordingly, the siRNAknockdown assay confirmed that the 6F9 TCR is MAGE-A3 specific.

Example 6

This example demonstrates that 6F9 TCR recognizes MAGE-A3 in an HLA-DPrestricted manner.

624, 526, 1359, H1299, 1300, 1764, 3071, 397, 2630, and 2984 tumor celllines were transduced with CIITA (624-CIITA, 526-CIITA, 1359-CIITA,H1299-CIITA, 1300-CIITA, 1764-CIITA, 3071-CIITA, 397-CIITA, 2630-CIITA,and 2984-CIITA) and HLA-DP expression was measured by flow cytometry.DP4 and MAGE-A3 expression is shown in Table 5A.

TABLE 5A Transduced Tumor Cell Line DP4 MAGE-A3 624-CIITA — + 1300-CIITA— + 3071-CIITA 0402 + Whitington-CIITA 0401 − 526-CIITA 0401 +1359-CIITA 0401 + H1299-CIITA 0401 + 397-CIITA 0401 + 2630-CIITA 0401 +2984-CIITA 0401 +

6F9-transduced PBL were cultured alone (T cells only) or co-culturedwith 3071 cells, 3071-CIITA cells, 397 cells, 397-CIITA cells, 2630cells, 2630-CIITA cells, 2984 cells, and 2984-CIITA cells. IFN-gammasecretion was measured. The results are shown in FIG. 3 . As shown inFIG. 3 , 6F9-transduced PBL were reactive with CIITA-expressing tumorcell lines.

The 6F9 TCR was further evaluated by determining the reactivity of CD4⁺and CD8⁺ T cells separated from two patient PBMCs against a panel oftumor cell lines including 624-CIITA, 526-CIITA, 1359-CIITA,H1299-CIITA, SK37-CIITA, 1764-CIITA, 3071-CIITA, 397-CIITA, 2630-CIITA,and 2984-CIITA. Five melanoma cell lines that expressed MAGE-A3 andHLA-DP*0401 (2630-CIITA, 397-CIITA, 2984-CIITA, 526-CIITA, and1359-CIITA), as well as the non-small cell lung carcinoma cell lineH1299 NSCLC-CIITA were recognized by transduced CD4⁺ and CD8⁺ T cells,although CD4⁺ T cells secreted higher amounts of cytokine in response totumor targets than transduced CD8⁺ T cells.

H1299-CIITA and 526-CIITA cells were transfected with anti-HLA-DP oranti-HLA-DR siRNA to knock down HLA-DP or HLA-DR expression. 3071-CIITAand 526-CIITA cells were transfected with anti-HLA-DQ siRNA to knockdown HLA-DQ expression. HLA-DP, HLA-DR, or HLA-DQ knockout was confirmedby flow cytometry.

PBL from a human donor were enriched for CD4+ and the number of cellswas rapidly expanded on day 30. The cells were transduced with 6F9 TCRor untransduced. The cells were cultured alone (T cell only) orco-cultured with untreated H1299-CIITA cells, H1299-CIITA transfectedwith anti-HLA-DP or anti-HLA-DR siRNA, untreated 526-CIITA cells, or526-CIITA transfected with anti-HLA-DP or anti-HLA-DR siRNA. IFN-gammasecretion was measured. The results are shown in FIG. 4 . As shown inFIG. 4 , the anti-HLA-DP siRNA reduced the reactivity of the 6F9-TCRtransduced cells.

Further studies employing antibodies confirmed that the 6F9 TCRrecognizes MAGE-A3 in an HLA-class II restricted manner. PBL transducedwith 6F9 TCR were co-cultured with the cells set forth in Table 5B andblocked with the antibodies set forth in Table 5B. IFN-gamma wasmeasured, and the results are set forth in Table 5B.

TABLE 5B 6F9 TCR-transduced PBL IFN-gamma co-cultured with: Blocked withantibody: (pg/ml) 293-CIITA (DP4+) W6/32 (α-HLA class I) >10,000transfected with MAGE-A3 HB22 (α-HLA class DR) >10,000 gene IVA12 (α-HLAclass II) 902 Allen B cells (A2+ DP4+) W6/32 (α-HLA class I) 15038Incubated with MAGE-A3 HB22 (α-HLA class DR) 16599 protein IVA12 (α-HLAclass II) 129 SK37 CIITA (A2+ DP4+ W6/32 (α-HLA class I) 1965 MAGE-A3+)HB22 (α-HLA class DR) 6248 IVA12 (α-HLA class II) 674 HI 299 CIITA (A2-DP4+ W6/32 (α-HLA class I) 2684 MAGE-A3+) HB22 (α-HLA class DR) 7888IVA12 (α-HLA class II) 0 1764 RCC CIITA (A2-DP4+ W6/32 (α-HLA class I) 0MAGE-A3-) HB22 (α-HLA class DR) 0 IVA12 (α-HLA class II) 0

As shown in Table 5B, the antibody blocking studies showed that the 6F9TCR recognizes MAGE-A3 in an HLA Class II-restricted manner, but not inan HLA-DR-restricted manner or in an HLA Class I-restricted manner.

Example 7

This example demonstrates that an alanine substitution at position 116or 117 of the alpha chain of the 6F9 TCR increases the reactivity of the6F9 TCR.

Eight different substituted TCRs, each having one alanine substitutionat a different location in the CDR3 region of the 6F9 TCR, were preparedas set forth in Table 6.

TABLE 6 Name Description SEQ ID NO: a1 Alanine substitution at positionSEQ ID NO: 12 (wild-type (wt) beta chain) 116 of alpha chain (S116A) SEQID NO: 33 (substituted alpha chain), wherein Xaa at 116 is Ala, Xaa at117 is Ser, Xaa at 118 is Gly, and Xaa at 119 is Thr a2 Alaninesubstitution at position SEQ ID NO: 12 (wild-type (wt) beta chain) 117of alpha chain (S117A) SEQ ID NO: 33 (substituted alpha chain), whereinXaa at 116 is Ser, Xaa at 117 is Ala, Xaa at 118 is Gly, and Xaa at 119is Thr a3 Alanine substitution at position SEQ ID NO: 12 (wild-type (wt)beta chain) 118 of alpha chain (G118A) SEQ ID NO: 33 (substituted alphachain), wherein Xaa at 116 is Ser, Xaa at 117 is Ser, Xaa at 118 is Ala,and Xaa at 119 is Thr a4 Alanine substitution at position SEQ ID NO: 12(wild-type (wt) beta chain) 119 of alpha chain (T119A) SEQ ID NO: 33(substituted alpha chain), wherein Xaa at 116 is Ser, Xaa at 117 is Ser,Xaa at 118 is Gly, and Xaa at 119 is Ala b1 Alanine substitution atposition SEQ ID NO: 11 (wild-type (wt) alpha chain) and 115 of betachain (R115A) SEQ ID NO: 34, wherein Xaa at 115 is Ala, Xaa at 116 isThr, Xaa at 117 is Gly, and Xaa at 118 is Pro b2 Alanine substitution atposition SEQ ID NO: 11 (wild-type (wt) alpha chain) and 116 of betachain (T116A) SEQ ID NO: 34, wherein Xaa at 115 is Arg, Xaa at 116 isAla, Xaa at 117 is Gly, and Xaa at 118 is Pro b3 Alanine substitution atposition SEQ ID NO: 11 (wild-type (wt) alpha chain) and 117 of betachain (G117A) SEQ ID NO: 34, wherein Xaa at 115 is Arg, Xaa at 116 isThr, Xaa at 117 is Ala, and Xaa at 118 is Pro b4 Alanine substitution atposition SEQ ID NO: 11 (wild-type (wt) alpha chain) and 118 of betachain (P118A) SEQ ID NO: 34, wherein Xaa at 115 is Arg, Xaa at 116 isThr, Xaa at 117 is Gly, and Xaa at 118 is Ala

PBL from a human donor were untransduced or transduced with wild-type(wt) 6F9 TCR or one of each of the eight substituted TCRs in Table 6.The cells were cultured alone (T cell only) or co-cultured with624-CIITA, 526-CIITA, 1359-CIITA, H1299-CIITA, or 1764-CIITA. IFN-gammasecretion was measured. The results are set forth in FIG. 5 . As shownin FIG. 5 , the a1 and a2 substituted TCRs demonstrated increasedreactivity as compared to wt 6F9 TCR.

A separate experiment with transduced, CD4+ enriched PBL also confirmedthe superior reactivity of the a1 and a2 substituted TCRs (FIG. 6 ). Asshown in FIG. 6 , the a1 and a2 substituted TCRs showed an approximately2-fold increase in anti-tumor activity as compared to wt 6F9 TCR. The a1and a2 substituted TCRs also showed better tetramer (SEQ ID NO: 2)binding as compared to the wt 6F9 TCR, as measured by flow cytometry.

Example 8

This example demonstrates the reactivity of substituted 6F9 TCRs.

Eight different substituted TCRs, each having one amino acidsubstitution at a different location in the CDR3 region of the alphachain of the 6F9 TCR, were prepared as set forth in Table 7.

TABLE 7 Name Description SEQ ID NO: a1-1 Leucine substitution atposition SEQ ID NO: 12 (wild-type (wt) beta chain) 116 of alpha chain(S116L) SEQ ID NO: 33 (substituted alpha chain), wherein Xaa at 116 isLeu, Xaa at 117 is Ser, Xaa at 118 is Gly, and Xaa at 119 is Thr a1-2Isoleucine substitution at position SEQ ID NO: 12 (wild-type (wt) betachain) 116 of alpha chain (S1161) SEQ ID NO: 33 (substituted alphachain), wherein Xaa at 116 is Ile, Xaa at 117 is Ser, Xaa at 118 is Gly,and Xaa at 119 is Thr a1-3 Valine substitution at position SEQ ID NO: 12(wild-type (wt) beta chain) 116 of alpha chain (S116V) SEQ ID NO: 33(substituted alpha chain), wherein Xaa at 116 is Val, Xaa at 117 is Ser,Xaa at 118 is Gly, and Xaa at 119 is Thr a1-4 Methionine substitution atposition SEQ ID NO: 12 (wild-type (wt) beta chain) 116 of alpha chain(S116M) SEQ ID NO: 33 (substituted alpha chain), wherein Xaa at 116 isMet, Xaa at 117 is Ser, Xaa at 118 is Gly, and Xaa at 119 is Thr a2-1Leucine substitution at position SEQ ID NO: 12 (wild-type (wt) betachain) 117 of beta chain (S117L) SEQ ID NO: 33 (substituted alphachain), wherein Xaa at 116 is Ser, Xaa at 117 is Leu, Xaa at 118 is Gly,and Xaa at 119 is Thr a2-2 Isoleucine substitution at position SEQ IDNO: 12 (wild-type (wt) beta chain) 117 of beta chain (S1171) SEQ ID NO:33 (substituted alpha chain), wherein Xaa at 116 is Ser, Xaa at 117 isIle, Xaa at 118 is Gly, and Xaa at 119 is Thr a2-3 Valine substitutionat position SEQ ID NO: 12 (wild-type (wt) beta chain) 117 of beta chain(S117V) SEQ ID NO: 33 (substituted alpha chain), wherein Xaa at 116 isSer, Xaa at 117 is Val, Xaa at 118 is Gly, and Xaa at 119 is Thr a2-4Methionine substitution at position SEQ ID NO: 12 (wild-type (wt) betachain) 117 of beta chain (S117M) SEQ ID NO: 33 (substituted alphachain), wherein Xaa at 116 is Ser, Xaa at 117 is Met, Xaa at 118 is Gly,and Xaa at 119 is Thr

PBL from a human donor were untransduced or transduced with wild-type(wt) 6F9 TCR or one of each of the eight substituted TCRs. The cellswere cultured alone (T cell only) or co-cultured with 624-CIITA,526-CIITA, 1359-CIITA, H1299-CIITA, or 1764-CIITA. IFN-gamma secretionwas measured. The results are set forth in FIG. 7 . As shown in FIG. 7 ,the a1, a2, and a1-3 substituted TCRs demonstrated reactivity againstCIITA-tumor cell lines.

Example 9

This example demonstrates that substitution of the native constantregion of the 6F9 TCR with a murine constant region increases thereactivity of the 6F9 TCR.

A TCR was prepared comprising the variable regions of the α and β chainsof the wt 6F9 TCR and a murine constant region (6F9mC TCR) (SEQ ID NOs:27 and 28).

The 6F9mC TCR demonstrated better MAGE-A3 tetramer and VP staining ascompared to wt 6F9 TCR, as measured by flow cytometry. Without beingbound to a particular theory, it is believed that the 6F9mC TCR providesimproved pairing of the TCR α and β chains.

PBL from a human donor were untransduced or transduced with wt 6F9 TCRor 6F9mC TCR and cultured alone (T-cell only) or co-cultured with624-CIITA, 1300-CIITA, 526-CIITA, 1359-CIITA, H1299-CIITA, 397-CIITA,2630-CIITA, 2984-CIITA, 3071-CIITA, or 1764-CIITA cells. IFN-gammasecretion was measured. The results are shown in FIG. 8 . As shown inFIG. 8 , the 6F9mC-transduced cells showed a 2-5 fold increase inanti-tumor activity as compared to wt 6F9 TCR-transduced cells.

Untransduced cells, 6F9 TCR-transduced cells, or 6F9mC TCR-transducedcells were enriched for CD8 or CD4 and cultured alone (T-cell only) orco-cultured with 624-CIITA, SK37-CIITA, 526-CIITA, 1359-CIITA,H1299-CIITA, 397-CIITA, 2630-CIITA, 2984-CIITA, 3071-CIITA, or1764-CIITA cells. Interferon-gamma secretion was measured. The resultsare shown in FIGS. 9A and 9B. As shown in FIGS. 9A and 9B, the CD8 andCD4 enriched 6F9mC-transduced cells maintained higher anti-tumoractivity as compared to 6F9 TCR transduced cells for several cell lines,indicating high affinity of the 6F9mC TCR independent of co-receptors.The experiments were repeated using PBL from a second human donor andsimilar results were obtained. Comparisons of responses of CD4+ T cellstransduced with the wild-type (Wt) 6F9 TCR with those of the cellstransduced with the 6F9mc TCR indicated that the murine constant regionsresulted in between two and five-fold enhancement in the response oftransduced T cells against the seven MAGE-A3⁺ and HLA-DP*0401⁺ targetsthat were evaluated. In addition, the response of CD8⁺ T cellstransduced with the 6F9mc were enhanced by between two and ten-foldabove those seen in cells transduced with the wt 6F9 TCR. The responsesof CD8⁺ T cells transduced with the 6F9mc were generally lower than CD4⁺T cells transduced with this TCR, although comparable cytokine responseswere observed in responses to some tumor targets.

Example 10

This example demonstrates that upon tumor stimulation, 6F9mCTCR-transduced cells produce high levels of IFN-gamma and TNF-alpha andshow a highly activated phenotype (as measured by increased 4-1BB, CD25,and CD69 expression).

Cells were CD4 or CD8 enriched and transduced with 6F9mC TCR. Transducedcells were co-cultured with tumor lines 624-CIITA, 2630-CIITA,2984-CIITA, or Whitington-CIITA for 6 hours and then stained forintracellular IFN-gamma, interleukin (IL)-2, or tumor necrosis factor(TNF)-α. The 6F9mC TCR transduced cells showed specific intracellularIFN-gamma production upon tumor stimulation. The 6F9mC TCR transducedcells showed detectable IL-2 production and specific high TNF-αproduction upon tumor stimulation in the CD4-enriched fraction.

Cells were CD4 enriched and transduced with 6F9mC TCR. Transduced cellswere co-cultured with tumor lines 624-CIITA, 2630-CIITA, 2984-CIITA, orWhitington-CIITA overnight and then stained for 4-1BB, CD25, and CD69.After overnight tumor stimulation, the majority of 6F9mC TCR-transducedcells expressed high levels of 4-1BB (indicative of antigen-specificactivation), CD25, and CD69.

Example 11

This example demonstrates that the 6F9 TCR mediates tumor cellrecognition.

PBL were untransduced or transduced with wild-type 6F9 TCR and culturedalone or co-cultured with non-small cell lung cancer (NSCLC) cell lineH11299 or melanoma cell line 526 mel, 624 mel, or 1359 mel. MAGE-A3 andDP*04 expression is shown in Table 8.

TABLE 8 Cell line MAGE-A3 DP*04 H1299 NSCLC + +  526 mel + +  624 mel +− 1359 mel + −

IFN-gamma expression was measured. The results are shown in FIG. 10B. Asshown in FIG. 10B, the 6F9 TCR mediates tumor cell recognition.

Example 12

This example demonstrates that the 6F9 and 6F9mc TCR possess a highdegree of specificity for the MAGE-A3:₂₄₈₋₂₅₈ peptide.

In order to evaluate the fine specificity of antigen recognitionmediated by cells transduced with the 6F9 and 6F9mc TCR, HLA-DP*0401⁺target cells were pulsed with truncations of the MAGE-A3:₂₄₃₋₂₅₈ peptideor related peptides from MAGE family members. CD4+ T cells isolated fromtwo patients' PBMC (PBMC-1 or PBMC-2) by negative selection weretransduced with either the 6F9 TCR, the 6F9mc TCR, or were un-transducedand assayed 10 days following OKT3 stimulation for their response to293-CIITA cells that were pulsed with 10 mg/ml of the peptides indicatedin Table 9.

Analysis of the response to truncated MAGE-A3 peptides from two culturesof transduced CD4⁺ PBMC indicated that the 11-mer peptide QHFVQENYLEY(SEQ ID NO: 54) corresponding to amino acids 248-258 of the MAGE-A3protein represented the minimal peptide that elicited a responsecomparable to that elicited by the MAGE-A3:₂₄₃₋₂₅₈ peptide (Table 9).The MAGE-A3:₂₄₃₋₂₅₈ peptide was predicted using an epitope predictionalgorithm to possess a high affinity for HLA-DP*0401, and in addition,recognition of the truncated MAGE-A3 peptides appeared to correlate withT cell recognition (Table 9). Significant recognition was observed forthe MAGE-A6:₂₄₈₋₂₅₈ peptide that contained a single substitution oftyrosine for histidine at position 249, but minimal reactivity wasobserved against additional members of the MAGE family of gene productsthat possessed between two and five differences from the MAGE-A3:₂₄₈₋₂₅₈peptide. A BLAST search of the NCBI database revealed that the mostclosely related peptide was derived from the protein necdin. Thispeptide, which possessed five differences from the MAGE-A3:₂₄₈₋₂₅₈peptide, was also not recognized by T cells transduced with the 6F9 or6F9mc TCR. These findings indicate that the 6F9 TCR possesses a highdegree of specificity for the MAGE-A3:₂₄₈₋₂₅₈ peptide, and suggest thatT cells transduced with this TCR may possess little or nocross-reactivity with peptides derived from additional human proteins.

TABLE 9 PBMC-1 PBMC-2 transduced with: transduced with: Predicted SEQAmino None 6F9 affinity Gene (position) ID NO: Acid Sequence 6F9 6F9mcIFN-g (pg/ml) 6F9mc None (nm) MAGE-A3: 243-258  2 KKLLTQHFVQENYLEY10,220 15,210 33 10,350 17,520  45    3 MAGE-A3: 243-256 47KKLLTQHFVQENYL  1,018  1,815 72  1,670 2,490  78  323 MAGE-A3: 243-25548 KKLLTQHFVQENY     76    137 29    111    117  71  378MAGE-A3: 243-254 49 KKLLTQHFVQEN     28     0 67     30     39  78  466MAGE-A3: 243-253 50 KKLLTQHFVQE      0     40 38     30     45  90 2444MAGE-A3: 245-258 51 LLTQHFVQENYLEY  9,290 14,970 84  8,920 17,820  74   3 MAGE-A3: 246-258 52 LTQHFVQENYLEY  7,140 12,700 56  9,200 16,170 76    3 MAGE-A3: 247-258 53 TQHFVQENYLEY  6,710 10,600 30  6,810 13,280 41    3 MAGE-A3: 248-258 54 QHFVQENYLEY  6,220  9,000 52  7,400  8,700 56    4 MAGE-A3: 249-258 55 HFVQENYLEY    669  1,643 57    922  2,034 66    5 MAGE-A6: 248-258 56 QYFVQENYLEY  6,440 11,800 54 13,200  8,370127    3 MAGE-A2/A12: 248-258 57 QDLVQENYLEY     33     66 49     37    56  65   59 MAGE-A4/A9: 249-259 58 QDWVQENYLEY      0     23 32    22     26  62   92 MAGE-A8: 251-261 59 QEWVQENYLEY     43     58 79    39     41  55   87 MAGE-A1/B4: 241-251 60 QDLVQEKYLEY    129    12655    108     84  53   16 MAGE-B2: 250-260 61 KDLVQEKYLEY     0      043      7     20  38   16 MAGE-B10: 250-260 62 KDLVKENYLEY     22     1869     28     34  66  105 MAGE-B16: 252-262 63 KDFVKEKYLEY      0     2716     11     28  42    3 MAGE-C1: 113-123 64 KVWVQEHYLEY      9      030     25     27  30   35 MAGE-D4: 300-315 65 RKLITDDFVKQKYLEY    193   234 81    194    268  80    6 MAGE-D2: 413-428 66 KKLITDEFVKQKYLDY    82     43 56    226    223  71    8 MAGE-L2: 582-597 67KKLITEVFVRQKYLEY     45     56 58     78    107 114    6MAGE-G1: 220-235 68 KKLITEDFVRQRYLEY      0     29 68     25     33  62   3 Necdin: 237-247 69 EEFVQMNYLKY      0     22 59     21     32  83  13 No peptide      0      5 58     15     25  59

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. (canceled)
 2. A method of producing an engineered population of humancells, the method comprising: introducing a recombinant expressionvector to an isolated population of human cells, wherein the recombinantexpression vector comprises a nucleotide sequence encoding a T-cellreceptor (TCR) having antigenic specificity for MAGE-A3₂₄₃₋₂₅₈ andMAGE-A6, wherein the TCR comprises the amino acid sequences of: (a) SEQID NOs: 3, 4, 6, 7, (i) SEQ ID NO: 29, wherein: Xaa4 is Ser, Ala, Leu,Ile, Val, or Met; Xaa5 is Ser, Ala, Leu, Ile, Val, or Met; Xaa6 is Gly,Ala, Leu, Ile, Val, or Met; and Xaa7 is Thr, Ala, Leu, Ile, Val, or Met;and (ii) SEQ ID NO: 30, wherein: Xaa4 is Arg, Ala, Leu, Ile, Val, orMet; Xaa5 is Thr, Ala, Leu, Ile, Val, or Met; Xaa6 is Gly, Ala, Leu,Ile, Val, or Met; and Xaa7 is Pro, Ala, Leu, Ile, Val, or Met; or (b)SEQ ID NOs: 3-8.
 3. The method according to claim 2, wherein the TCRcomprises the amino acid sequences of SEQ ID NOs: 3-8.
 4. The methodaccording to claim 2, wherein the TCR comprises a murine constantregion.
 5. The method according to claim 4, wherein the TCR comprisesthe murine constant region amino acid sequence(s) of SEQ ID NO: 25and/or SEQ ID NO:
 26. 6. The method according to claim 2, wherein theTCR comprises: a first amino acid sequence selected from the groupconsisting of: (i) SEQ ID NO: 31, wherein: Xaa116 is Ser, Ala, Leu, Ile,Val, or Met; Xaa117 is Ser, Ala, Leu, Ile, Val, or Met; Xaa118 is Gly,Ala, Leu, Ile, Val, or Met; and Xaa119 is Thr, Ala, Leu, Ile, Val, orMet; and (ii) SEQ ID NO: 9; and a second amino acid sequence selectedfrom the group consisting of: (i) SEQ ID NO: 32, wherein: Xaa115 is Arg,Ala, Leu, Ile, Val, or Met; Xaa116 is Thr, Ala, Leu, Ile, Val, or Met;Xaa117 is Gly, Ala, Leu, Ile, Val, or Met; and Xaa118 is Pro, Ala, Leu,Ile, Val, or Met; and (ii) SEQ ID NO:
 10. 7. The method according toclaim 6, wherein the TCR comprises the amino acid sequences of SEQ IDNO: 9 and SEQ ID NO:
 10. 8. The method according to claim 2, wherein theTCR comprises: a first amino acid sequence selected from the groupconsisting of: (i) SEQ ID NO: 33, wherein: Xaa116 is Ser, Ala, Leu, Ile,Val, or Met; Xaa117 is Ser, Ala, Leu, Ile, Val, or Met; Xaa118 is Gly,Ala, Leu, Ile, Val, or Met; and Xaa119 is Thr, Ala, Leu, Ile, Val, orMet; (ii) SEQ ID NO: 11; and (iii) SEQ ID NO: 27; and a second aminoacid sequence selected from the group consisting of: (i) SEQ ID NO: 34,wherein: Xaa115 is Arg, Ala, Leu, Ile, Val, or Met; Xaa116 is Thr, Ala,Leu, Ile, Val, or Met; Xaa117 is Gly, Ala, Leu, Ile, Val, or Met; andXaa118 is Pro, Ala, Leu, Ile, Val, or Met; (ii) SEQ ID NO: 12; and (iii)SEQ ID NO:
 28. 9. A method of producing an engineered population ofhuman cells, the method comprising: introducing a recombinant expressionvector to an isolated population of human cells, wherein the recombinantexpression vector comprises a nucleotide sequence encoding a polypeptidecomprising the amino acid sequences of: (a) SEQ ID NOs: 3, 4, 6, 7, (i)SEQ ID NO: 29, wherein: Xaa4 is Ser, Ala, Leu, Ile, Val, or Met; Xaa5 isSer, Ala, Leu, Ile, Val, or Met; Xaa6 is Gly, Ala, Leu, Ile, Val, orMet; and Xaa7 is Thr, Ala, Leu, Ile, Val, or Met; and (ii) SEQ ID NO:30, wherein: Xaa4 is Arg, Ala, Leu, Ile, Val, or Met; Xaa5 is Thr, Ala,Leu, Ile, Val, or Met; Xaa6 is Gly, Ala, Leu, Ile, Val, or Met; and Xaa7is Pro, Ala, Leu, Ile, Val, or Met; or (b) SEQ ID NOs: 3-8.
 10. Themethod of claim 9, wherein the polypeptide comprises the amino acidsequences of SEQ ID NOs 3-8.
 11. The method of claim 9, wherein thepolypeptide comprises: a first amino acid sequence selected from thegroup consisting of: (i) SEQ ID NO: 31, wherein: Xaa116 is Ser, Ala,Leu, Ile, Val, or Met; Xaa117 is Ser, Ala, Leu, Ile, Val, or Met; Xaa118is Gly, Ala, Leu, Ile, Val, or Met; and Xaa119 is Thr, Ala, Leu, Ile,Val, or Met; and (ii) SEQ ID NO: 9; and a second amino acid sequenceselected from the group consisting of: (i) SEQ ID NO: 32, wherein:Xaa115 is Arg, Ala, Leu, Ile, Val, or Met; Xaa116 is Thr, Ala, Leu, Ile,Val, or Met; Xaa117 is Gly, Ala, Leu, Ile, Val, or Met; and Xaa118 isPro, Ala, Leu, Ile, Val, or Met; and (ii) SEQ ID NO:
 10. 12. The methodof claim 11, wherein the polypeptide comprises the amino acid sequencesof SEQ ID NO: 9 and SEQ ID NO:
 10. 13. The method of claim 9, whereinthe polypeptide comprises: a first amino acid sequence selected from thegroup consisting of: (i) SEQ ID NO: 33, wherein: Xaa116 is Ser, Ala,Leu, Ile, Val, or Met; Xaa117 is Ser, Ala, Leu, Ile, Val, or Met; Xaa118is Gly, Ala, Leu, Ile, Val, or Met; and Xaa119 is Thr, Ala, Leu, Ile,Val, or Met; (ii) SEQ ID NO: 11; and (iii) SEQ ID NO: 27; and a secondamino acid sequence selected from the group consisting of: (i) SEQ IDNO: 34, wherein: Xaa115 is Arg, Ala, Leu, Ile, Val, or Met; Xaa116 isThr, Ala, Leu, Ile, Val, or Met; Xaa117 is Gly, Ala, Leu, Ile, Val, orMet; and Xaa118 is Pro, Ala, Leu, Ile, Val, or Met; (ii) SEQ ID NO: 12;and (iii) SEQ ID NO:
 28. 14. A method of producing an engineeredpopulation of human cells, the method comprising: introducing arecombinant expression vector to an isolated population of human cells,wherein the recombinant expression vector comprises a nucleotidesequence encoding a protein comprising a first polypeptide chaincomprising the amino acid sequences of: (a) SEQ ID NO: 3-5; or (b) SEQID NOs: 3, 4, and SEQ ID NO: 29, wherein: Xaa4 is Ser, Ala, Leu, Ile,Val, or Met; Xaa5 is Ser, Ala, Leu, Ile, Val, or Met; Xaa6 is Gly, Ala,Leu, Ile, Val, or Met; and Xaa7 is Thr, Ala, Leu, Ile, Val, or Met; anda second polypeptide chain comprising the amino acid sequences of: (a)SEQ ID NOs: 6-8; or (b) SEQ ID NOs: 6, 7, and SEQ ID NO: 30, wherein:Xaa4 is Arg, Ala, Leu, Ile, Val, or Met; Xaa5 is Thr, Ala, Leu, Ile,Val, or Met; Xaa6 is Gly, Ala, Leu, Ile, Val, or Met; and Xaa7 is Pro,Ala, Leu, Ile, Val, or Met.
 15. The method of claim 14, wherein thefirst polypeptide chain comprises the amino acid sequences of SEQ ID NO:3-5 and the second polypeptide chain comprises the amino acid sequencesof SEQ ID NO: 6-8.
 16. The method according to claim 14, wherein thefirst polypeptide chain comprises an amino acid sequence selected fromthe group consisting of: (i) SEQ ID NO: 31, wherein: Xaa116 is Ser, Ala,Leu, Ile, Val, or Met; Xaa117 is Ser, Ala, Leu, Ile, Val, or Met; Xaa118is Gly, Ala, Leu, Ile, Val, or Met; and Xaa119 is Thr, Ala, Leu, Ile,Val, or Met; and (ii) SEQ ID NO: 9; and the second polypeptide chaincomprises an amino acid sequence selected from the group consisting of:(i) SEQ ID NO: 32, wherein: Xaa115 is Arg, Ala, Leu, Ile, Val, or Met;Xaa116 is Thr, Ala, Leu, Ile, Val, or Met; Xaa117 is Gly, Ala, Leu, Ile,Val, or Met; and Xaa118 is Pro, Ala, Leu, Ile, Val, or Met; and (ii) SEQID NO:
 10. 17. The method of claim 16, wherein the first polypeptidechain comprises the amino acid sequence of SEQ ID NO: 9 and the secondpolypeptide chain comprises the amino acid sequence of SEQ ID NO: 10.18. The method of claim 14, wherein the first polypeptide chaincomprises an amino acid sequence selected from the group consisting of:(i) SEQ ID NO: 33, wherein: Xaa116 is Ser, Ala, Leu, Ile, Val, or Met;Xaa117 is Ser, Ala, Leu, Ile, Val, or Met; Xaa118 is Gly, Ala, Leu, Ile,Val, or Met; and Xaa119 is Thr, Ala, Leu, Ile, Val, or Met; (ii) SEQ IDNO: 11; and (iii) SEQ ID NO: 27; and the second polypeptide chaincomprises an amino acid sequence selected from the group consisting of:(i) SEQ ID NO: 34, wherein: Xaa115 is Arg, Ala, Leu, Ile, Val, or Met;Xaa116 is Thr, Ala, Leu, Ile, Val, or Met; Xaa117 is Gly, Ala, Leu, Ile,Val, or Met; and Xaa118 is Pro, Ala, Leu, Ile, Val, or Met; (ii) SEQ IDNO: 12; and (iii) SEQ ID NO:
 28. 19. The method of claim 2, wherein theisolated population of human cells is an isolated population of humanperipheral blood lymphocytes or an isolated population of humanperipheral blood mononuclear cells.
 20. The method of claim 2, whereinthe isolated population of human cells is an isolated population ofhuman T cells.
 21. The method of claim 2, wherein the isolatedpopulation of human cells is an isolated population of human CD4⁺ Tcells.